1TOLOGY-I 


BIOLOGY 

LIBRARY 

G 


A   COURSE 

IN 

NORMAL    HISTOLOGY 


PUBLISHERS'  ANNOUNCEMENT 


RUDOLPH  KRAUSE— HISTOLOGY 

We  offer  this  book  to  teachers  and  students  alike  as  a  proper  guide 
in  Histology. 

Professor  Krause  strikes  a  keynote  in  his  preface  to  the  book.  The 
study  of  Histology  should  go  hand  in  hand  with  that  of  Anatomy.  The 
one  cannot  be  separated  from  the  other  nowadays  without  serious  injury 
to  the  student. 

The  microscope  is  the  hand-maid  of  anatomy.  For  this  reason  it  has 
been  deemed  expedient  to  publish  the  book  in  two  separate  sections. 

The  first  part  is  simply  a  guide  to  the  technique  of  microscopy,  and 
may  be  used  by  students  of  medicine  as  well  as  by  those  who  pursue  sub- 
jects of  science  foreign  to  medicine. 

The  second  part  deals  exclusively  with  histology  appertaining  to  medi- 
cine. Either  part  may  be  obtained  independent  of  the  other. 

In  bringing  this  work  before  the  public  it  is  the  author's  aim  to  give 
to  the  student  a  book  of  reference  which  is  both  practical  and  theoretical, 
as  well  as  to  furnish  the  teacher  with  a  text-book  which  provides  him  with 
a  detailed,  yet  clear,  concise  and  methodical  guide  through  the  course  of 
Microscopy  and  Histology. 

So  far  as  the  student  of  medicine  is  concerned,  he  will  find  therein  much 
valuable  information  appertaining  to  the  microtechnique  which  must  neces- 
sarily be  taught  to  the  exclusive  student  of  Pathology.  The  subject  of 
microtechnique  is  in  most  all  English-written  books  on  this  topic  treated 
but  very  briefly.  This  work  not  only  teaches  the  student,  in  a  most  thor- 
ough fashion,  the  theory  and  manipulation  of  the  microscope,  but  enables 
him  to  acquaint  himself  with  all  the  methods  employed  in  preparing  a  spec- 
imen for  microscopical  examination  from  start  to  finish. 

REBMAN    COMPANY. 

NEW  YORK,  1123  Broadway. 


A  COURSE 

IN 

NORMAL  HISTOLOGY 

A    GUIDE   FOR 

PRACTICAL  INSTRUCTION  IN  HISTOLOGY 
AND  MICROSCOPIC  ANATOMY 

BY 

RUDOLF    KRAUSE 

A.   O.    PROFESSOR  OF  ANATOMY  AT  THE  UNIVERSITY  OF  BERLIN 
TRANSLATION    FROM   THE   GERMAN    BY 

PHILIPP  J.    R.    SCHMAHL,    M.D. 

NEW    YORK 


WITH  30  ILLUSTRATIONS  IN  TEXT  AND  208   COLORED  PICTURES, 

ARRANGED   ON  98  PLATES  AFTER   THE  ORIGINAL 

DRAWINGS  BY  THE  AUTHOR 

PART    I. 


NEW  YORK 
REBMAN  COMPANY 

1123     BROADWAY 


. 


B1OLOQV 
LIBRARY 

G 


COPYRIGHT,  1913,  BY 

REBMAN    COMPANY 

NEW  YORK 

All  Rights  reserved 


PRINTED  IN  AMERICA 


DEDICATED 

TO 

PROFESSOR  DR.  WILHELM  WALDEYER 

SEC.    MED.   COUNCILLOR 

IN     GRATEFUL     ESTEEM 
BY  THE   AUTHOR 


Preface 

The  brilliant  progress  made  in  microscopy  during  the  last  three  to  four 
decades  has  gradually  brought  with  it  a  total  change  in  the  course  of  histologic 
instruction.  In  former  days  the  student  himself  prepared  his  own  microscopic 
specimens  with  razor  and  needle  and  with  the  aid  of  a  few  solutions  for  macera- 
tion, hardening  and  staining.  To-day,  after  recognizing  the  inefficiency  of  such 
methods,  this  work  has  almost  entirely  been  taken  from  the  student's  hands. 
Instead  he  receives  from  the  assistant  or  preparator  more  or  less  immaculate 
specimens,  preserved,  cut  and  stained;  some  only  for  a  single  observation^ 
others  as  his  property.  The  practical  course  has  been  replaced  by  one  of 
demonstration ;  at  any  rate  in  all  its  essential  phases. 

Special  courses  have  been  designed  for  instruction  in  the  technique  of 
microscopy,  with  the  result  that  such  courses  have  but  a  minimum  percentage 
of  attendance.  By  far  the  majority  of  our  students  enter  the  clinical  semesters 
without  being  able  to  prepare  a  useful  microscopic  specimen.  Not  until  later, 
during  a  course  in  pathologic  histology  or  in  the  laboratories  of  clinics,  will 
they  learn  those  micro-technical  manipulations  so  essential  to  them  as  prac- 
titioners. 

This,  in  my  estimation,  is  not  a  normal  state  of  affairs.  Every  medical 
student,  upon  entering  the  clinical  semesters,  should  be  able  to  master  micro- 
technical  methods  to  such  a  degree  that  he  is  able  to  prepare  £,  good  specimen 
for  microscopic  examination  from  any  organ  given  to  him.  This  will  be  possi- 
ble only  when  the  course  in  histology  occupies  that  rank  of  which  it  is  deserv- 
ing; that  is  to  say,  when  the  student  ceases  to  be  granted  the  meagre  allowance 
of  only  four,  at  the  utmost  six  hours  per  week;  but,  on  the  contrary,  as  has 
already  been  attempted  in  several  places,  when  he  is  permitted  to  make  use  of 
one  entire  summer  semester  to  such  advantage  as  was  proffered  heretofore  dur- 
ing two  preparatory  winter  semesters.  Then,  such  courses  as  "Technical  In- 
struction," "Advanced  Course,"  or  whatever  they  may  be  called,  will  be  dis- 
pensed with.  The  average  medical  man  must  receive  an  adequate  technical 
education  in  histology.  The  scientist  should  seek  the  laboratory. 

During  the  time  that  the  student  does  practical  histologic  work,  he  should 
be  bound  to  improve  his  theoretical  knowledge  also,  which  at  the  very  best,, 
those  who  know  will  admit,  leaves  much  room  for  improvement  under  present 
conditions  Without  doubt  the  student  takes  a  greater  interest  in  a  specimen 

vi 


Vll 


made  by  himself  than  in  one  already  prepared,  whether  presented  to  him  or 
only  loaned. 

The  aim  of  this  book  is  to  direct  the  student  to  a  practical  education  in 
histology  and  to  microscopic  anatomy,  by  the  simplest  methods.  Most  of  the 
specimens  described  can  be  prepared  from  beginning  to  end  by  the  student  him- 
self, if  he  employs  the  technique  of  freezing,  which  has  long  since  acquired  the 
title  of  "standard"  in  pathologic  institutions,  but  was  wofully  neglected  by 
anatomists  in  the  past. 

By  dividing  a  larger  class  of  students  into  separate  divisions,  each  one  of 
which  being  supplied  with  a  freezing  microtome,  every  participant  will  find 
ample  opportunity  during  the  summer  period  to  attain  experience  in  microt- 
omy. Furthermore,  it  will  be  possible  for  every  student  of  each  single  group 
to  go  through  all  the  methods  of  embedding  in  paraffin  and  celloidin  at  least 
once,  from  beginning  to  end,  during  the  semester.  Moreover,  it  must  be  under- 
stood that  unstained  paraffin  sections  are  given  out  on  isinglass.  The  staining 
must  be  done  in  all  cases  by  the  student  himself,  whether  frozen,  paraffin  or 
celloidin  sections  are  used. 

It  goes  without  saying  that  staining  of  living  tissues  with  methylene  blue, 
injection  of  blood-vessels  and  lymph-ducts,  and  similar  complicated  technical 
manipulations  cannot  at  once  be  performed  by  the  student  himself.  He  will 
learn  by  witnessing  these  procedures.  And  this,  too,  can  be  easily  accomplished 
when  the  classes  are  grouped  into  subdivisions. 

I  assume,  against  custom,  that  the  student  is  supplied  with  a  modern  micro- 
scope having  a  condenser  and  homogeneous  immersion.  The  latter,  of  course, 
may  be  replaced  by  high  power  dry  system  to  some  extent.  I  hold  that  what 
has  been  an  established  factor  in  the  bacteriological  course  hitherto,  should  also 
be  made  possible  in  histology.  When  we  look  at  the  time-honored  Schicks, 
Ploessls,  Hartnacks,  etc.,  employed  in  some  of  the  histologic  courses,  we  cannot 
help  but  notice  that  we  have  not  kept  in  step  with  other  lines  of  our  science. 
Granted  that  the  experienced  eye  can  see  most  things  well  enough  even  with  the 
aid  of  such  instruments,  we  should  not  add  to  the  difficulties  which  confront  the 
beginner,  the  additional  tax  of  inadequate  tools.  On  the  contrary  we  should 
seek  to  assist  him  in  his  studies  by  placing  at  his  disposal  the  most  perfect  in- 
struments. It  is  of  the  greatest  importance  to  use  the  best  drawings  that  can 
be  made  of  the  specimens,  and  every  student  should  be  obliged  to  make  faithful 
sketches  of  his  slides.  Microscopic  drawing  is  not  an  art  in  the  strict  sense 
of  the  word.  In  order  to  stimulate  drawing  by  students,  I  have  undertaken 
the  task  of  sketching  every  preparation  myself.  These  drawings,  no  more  than 
those  of  students,  are  not  intended  nor  expected  to  be  works  of  art,  but  solely 
true  representations  of  the  microscopic  findings.  Each  sketch  has  been  pre- 
pared with  the  aid  of  Abbe's  drawing  apparatus,  and  the  enlargement  is  marked 


Vlll 

on  every  one.  The  fraction,  which  is  often  annexed,  indicates  how  much  the 
original  has  been  reduced  in  the  reproduction. 

To  obtain  the  truest  image  possible  of  the  original  drawing,  I  have  em- 
ployed the  autotype,  which  has  attained  such  a  standard  of  perfection  to-day 
as  to  bring  out  the  finest  details  of  a  microscopic  image.  The  cuts  have  been 
made  in  a  most  excellent  manner  by  the  Anger er  and  Goeschl  Art  Printing  Co., 
of  Vienna.  The  printing  was  done  by  Christoph  Reisser's  Sons,  also  of  Vienna. 
To  both  firms  I  am  indebted  for  their  readiness  in  giving  my  wishes  the  closest 
attention,  and  also  for  the  superior  execution  of  the  plates. 

A  text  is  furnished  with  every  drawing.  It  precedes  each  plate,  explains 
the  technique,  and  gives  a  brief  description  of  the  specimen.  If  the  latter  is 
not  confined  entirely  in  all  the  details  to  the  drawing,  but  deals  also  with  other 
matters,  not  depicted,  it  does  not  follow  that  this  guide-book  pretends  to 
represent  a  text-book  of  histology,  which  it  cannot  be  any  more  than  the  his- 
tologic  course  can  be  an  adequate  substitute  for  a  course  of  lectures  in  his- 
tology and  microscopic  anatomy ;  they  supplement  one  another,  but  the  one 
cannot  replace  the  other.  For  that  reason  it  is  desirable  that  text-books  on 
histology  should  cast  off  that  technical  ballast  which  they  carry  in  contra- 
distinction to  other  departments. 

In  fine  I  deem  it  a  pleasurable  duty  to  heartily  thank  my  publishers.  They 
have  met  my  wishes  in  every  detail  and  have  spared  neither  pains  nor  money  in 
order  to  equip  this  volume  in  the  most  lavish  manner. 

BERLIN.  RUDOLF  KRAUSE. 


Translator's  Preface 

At  the  request  of  Mr.  F.  J.  Rebman,  I  have  undertaken  the  translation  of 
Rudolf  Krause's  Histology  into  the  English  language.  I  have  adhered  to  the 
German  text  as  closely  as  possible,  without,  however,  making  sacrifices  to 
idiomatic  expressions.  My  aim  has  been  to  make  the  book  as  useful  to  the 
student  of  medicine  in  English-speaking  countries,  as  is  compatible  with  the 
spirit  in  which  the  author  has  written  it. 

Professor  Krause  seeks  to  raise  the  level  of  instruction  in  Histology  to  that 
obtained  during  recent  years  in  other  departments  of  medical  education.  He 
deserves  encouragement  in  this  praiseworthy  endeavor,  and  the  authorities 
entrusted  with  the  education  of  aspirants  in  medicine  at  the  various  colleges 
are  supporting  him  in  his  claim. 

It  affords  me  pleasure,  therefore,  to  be  enabled  to  place  at  the  disposal  of 
teachers  and  students  of  Histology  an  English  version  of  Professor  Krause's 
teachings,  and  I  sincerely  trust  that  the  book  will  find  a  proper  place  in  the 
curriculum  of  the  medical  schools  of  all  countries  in  which  the  English  language 
is  spoken. 

I  take  pride  in  expressing  my  obligation  to  Mr.  Rebman  for  the  aid  lie  has 
given  me  in  preparing  my  manuscript,  especially  in  the  translation  of  the 
preface  by  the  author.  I  likewise  cheerfully  acknowledge  the  assistance  derived 
from  the  perusal  of  Edward  Bausch's  "Manipulation  of  the  Microscope." 

P.   J.   R.    SCHMAHL,   M.D. 

NEW  YORK  CITY. 


IX 


Table  of  Contents 


PAGE 

PREFACE vii 

TRANSLATOR'S   PREFACE    xi 

INTRODUCTION    1 

GENERAL  PART 3 

The    Microscope    5 

The  Objective   12 

The  Eyepiece    17 

Illumination    18 

The  Micrometer-Screw    21 

Rules  for  the  Use  of  the  Microscope 21 

General  Microtechnique    24 

Methods  of   Preservation    25 

Preparation   of    Sections 34 

Dissociation   Methods    34 

Chopping  Method     35 

Cutting  Methods    36 

(a)   Frozen  Section    37 

(6)   Paraffin  Section    41 

(c)   Celloidin  Section    46 

Selection  of  Method 47 

Appendix    48 

Staining   Methods    48 

Stains  of  Animal  Origin 54 

Stains  of  Vegetable  Origin 55 

Artificial    Stains     58 

Basic    Dyes    58 

Acid   Dyes    63 

Indifferent   Stains    65 

Staining  Mixtures    65 

Appendix   68 

Methods   of   Injection 73 

Mounting  and  Finishing 76 

Mensuration  and  Drawing 79 

x 


Introduction 


THE  AIM  OF  A   COURSE  IN  HISTOLOGY 

The  object  of  the  histologic  course  is  a  dual  one:  on  one  hand  it  should 
serve  as  a  completion  of  the  theoretical  lectures  on  general  and  systematized 
anatomy,  i.e.,  it  should  demonstrate  ad  oculos  what  the  student  has  been  told 
of  the  microscopic  structure  of  the  human  body  and  enable  him  to  understand, 
recognize  and  differentiate  for  himself  the  organs  and  the  tissues  composing 
them.  The  daily  practical  work  with  the  microscopic  preparation  should  give 
him  a  fixed  and  definite  idea  of  the  normal  structures  of  the  various  organs, 
in  order  to  enable  him  in  later  semesters  to  recognize  and  diagnose  the  patho- 
logic changes  found  therein. 

If  this  were  the  only  object,  it  would  suffice  to  assign  a  number  of  good 
specimens  to  the  student,  to  discuss  them  thoroughly  with  him,  and  to  let  him 
reproduce  them  with  the  pencil  on  paper.  But  such  is  not  the  case.  Our 
course  of  instruction  has  a  secondary  aim,  equally  important  and  a  good  deal 
more  practical,  viz.,  to  initiate  the  student  in  micro-technique,  which  latter 
is  of  the  greatest  importance  for  advanced  study  as  well  as  general  practical 
education.  At  the  present  day  the  microscope  is  probably  the  most  important 
and  necessary  requisite  of  a  medical  instrumentarium.  A  thorough  training 
is  needed  for  handling  it  in  the  proper  manner,  and  the  basis  of  this  training 
rests  with  the  histologic  course,  at  the  end  of  which  the  student  should  be 
thoroughly  familiar  with  the  microscope  and  its  use. 

But  this  is  by  no  means  sufficient.  The  use  of  the  microscope  presupposes 
the  knowledge  how  to  prepare  a  specimen  for  examination,  and  for  this  reason 
it  becomes  necessary  for  the  student  to  acquire  skill  in  what  is  commonly 
termed  microscopic  technique  or  micro-technique.  This,  however,  is 
an  extremely  large  field,  which  requires  years  of  study  to  master,  and  no  sane 
person  will  ask  the  student  to  familiarize  himself  with  all  the  innumerable 
methods  and  details  of  procedure.  Nevertheless  he  should  attain  that  amount 
of  technique  to  enable  him  to  prepare,  from  any  given  organ,  a  specimen  which 
clearly  shows  its  structural  peculiarities. 

To  be  a  guide  in  accomplishing  this  end  is  the  object  of  this  volume. 
Primarily  it  will  familiarize  the  reader  with  the  microscope  and  its  use.  Fur- 
thermore, it  will  bring  to  his  attention  the  most  important  reagents,  stains,  and 
microtechnical  methods;  and  lastly,  in  the  Special  Part  it  will  give  detailed 
instructions  for  the  production  of  good  specimens,  suitable  to  the  study  of  each 
tissue  and  organ,  and  to  elucidate  in  words  and  pictures  what  may  be  learned 
from  these  specimens. 

1 


Untieing  pains  have  been  taken  to  reproduce  the  most  characteristic 
parts  of  a  preparation.  The  cuts  should  stimulate  the  reader  to  draw  speci- 
mens for  himself.  If  the  first  endeavors  should  prove  unsatisfactory,  there  is 
hardly  anything  else  that  will  be  so  easily  and  quickly  learned  with  a  little 
perseverance  and  a  bit  of  adroitness,  than  is  microscopic  drawing,  which  does 
not,  as  the  beginner  generally  fears,  necessarily  require  any  special  talent. 
Not  until  drawing  it,  do  we  fully  appreciate  a  specimen  and  learn  all  about 
it,  and  our  drawing,  when  completed,  will  forever  remain  vivid  in  our  memory, 
an  indelible  picture. 


GENERAL  PART 


The  Microscope 


The  elements  constituting  the  human  and  animal  organism  are  of  such 
minute  size  that  in  most  instances  they  cannot  be  recognized  by  the  unaided 
eye ;  the  necessity  arises  therefore,  to  employ  certain  instruments  which  will 
present  to  our  eye  a  magnified  image.  The  knowledge  of  such  agents  dates  far 
back  in  history.  The  fact  that  lenses,  i.e.,  transparent  spherical  bodies,  will 
under  certain  conditions  magnify  the  articles  viewed  through  them,  has  been 
known  for  ages. 

Lenses  as  a  Means 
of  Magnifying. 

In  ancient  times  lenses  were  made  from  more  or  less  valuable  natural  crys- 
tals. This  is  on  rare  occasions  done  even  to  th|s  day ;  but  as  a  rule  lenses  are 
now  made  of  artificial  glass  of  a  fixed  chemical  combination,  for  it ;  has  been 
found  that  the  composition  has  a  great  influence  on  the  optic  properties,  i.e., 
on  refraction  and  dispersion  power.  Lenses  with  moderate  dispersion  and 
greater  refraction  power  are  designated  as  crown  glasses,  those  with  high 
dispersion  as  flint  glasses.  In  the  preparation  of  the  latter  a  large  per- 
centage of  lead  oxide  is  added  to  the  liquid  glass.  They  also  contain  arsenic, 
manganese,  and,  of  course,  silicon  dioxide  in  small  amounts.  Crown  glasses, 
on  the  other  hand,  contain  also  a  good  percentage  of  silicon  dioxide,  the  alkali 
metals,  alkaline  earth  metals  and  boric  acid  anhydride.  It  follows  that  flint 
glasses  are,  as  a  rule,  heavier  than  crown  glasses.  The  dispersion  power  of  the 
flint  is  in  direct  proportion  to  its  weight,  i.e.,  amount  of  lead  contained  in  it. 

Classification  of  Lenses. 

According  to  their  form  lenses  may  be  divided  into  two  main  divisions: 
those  which  are  thicker  in  their  centre,  the  optic  axis,  than  on  the  periphery, 
and  those  in  which  the  thickness  decreases  from  periphery  to  optic  axis.  The 


FIG.  1. 
Positive  Lenses. 


FIG.  2. 

Negative  Lenses. 


former  are  called  positive  or  convex  lenses,  while  the  latter  are  known  as 
negative  or  concave  lenses.  Under  the  positive  lenses  we  again  distinguish 
between  biconvex,  plano-convex  and  positive  meniscus  (Fig.  1).  All  parallel 

5 


6 

rays  falling  on  a  positive  lens  are  known  to  unite  in  a  point  on  the  other  side 
of  the  lens,  situated  on  the  optic  axis  and  called  the  focus.  The  distance  of 
the  focus  from  the  lens  is  termed  focal  distance.  Parallel  rays  falling  on  a 
negative  lens  are  dispersed  in  such  a  fashion  as  if  emanating  from  a  point  this 
side  of  the  glass.  Here  also  we  speak  of  focus  and  focal  distance,  but  prefix 
them  with  negative  or  virtual.  Among  the  negative  lenses  we  also  speak  of  bi- 
concave, plano-concave  and  negative  meniscus  (Fig.  2). 

Image-Formation  Through 
a  Biconvex  Lens, 

According  to  known  optic  laws  a  biconvex  lens  furnishes  an  inverted  real 
picture  of  an  object,  situated  beyond  its  focal  distance,  i.e.,  in  our  case  (Fig.  3) 


FIG.  3. 
Reproduction  through  a  Biconvex  Lens. 


FIG.  4. 
Reproduction  through  a  Biconcave  Lens. 


an  image  situated  to  the  right  of  the  lens  (a1  6X — A1  B^).  If  the  object 
approaches  the  focus,  the  picture  recedes  from  the  lens,  becoming  larger 
(a2  62 — A2  J52),  and  finally,  when  the  object  occupies  the  focus,  is  lost  ad 
infinitum.  If  the  object  encroaches  on  the  focal  distance  itself,  we  get  an  erect, 
enlarged,  virtual  image,  situated  to  the  left  of  the  lens  (a3  63 — Az  -B3).  The 
closer  the  object  approaches  the  lens  the  smaller  the  picture  will  be,  though 
always  remaining  larger  than  the  object  itself  («4  b4 — A4  B4). 


The  Biconcave  Lens. 

Much  simpler  is  the  action  of  a  biconcave  lens.  It  gives  us  under  all  con- 
ditions a  reduced  erect  image,  which  becomes  larger  as  the  object  is  approxi- 
mated to  the  lens  (Fig.  4). 

Magnifying. 

If  we  want  to  distinctly  perceive  with  our  eyes  a  picture,  reflected  by  a 
lens,  it  becomes  necessary  to  establish  a  distance  between  image  and  eye  of 
approximately  250  mm,  i.e.,  to  place  it  in  distinct  visual  distance.  The  mag- 
nifying power  furnished  by  a  certain  lens  is  dependent  upon  its  focal  dis- 
tance and  is  found  by  dividing  the  distinct  visual  distance  of  the  particular 
observer  by  the  focal  distance,  both  expressed  in  millimetres.  The  focal  dis- 
tance in  turn  is  dependent  upon  the  curvature  of  the 
planes  of  the  lens  and  the  refraction  index  of  the  par-  /TV  7\ 

ticular  glass.    For  example,  we  find  a  focal  distance  of  the          \\  \ 

lenses,  represented  in  Fig.  1,  of  50  mm  for  the  biconvex, 
100  mm  for  the  plano-convex  and  150  mm  for  the  menis-- 
cus,  presuming  the  refraction  index  to  be  1.5.  These 
lenses,  therefore,  would  possess  a  magnifying  power  of 
5,  2.5  and  1.6  respectively.  With  the  negative  lenses  of 
Fig.  2,  on  the  other  hand,  we  get  a  focal  distance  of  — 50 
mm  for  the  biconcave,  — 100  mm  for  the  plano-concave 
and  — 150  mm  for  the  meniscus.  These  lenses,  therefore,  pIG  5 

will  reduce  correspondingly.  Compound  Lenses. 

Very  often  two  or  more  lenses  are  combined  and  ce- 
mented together  by  a  transparent  resin  in  a  fashion  that  they  are  intimately 
approximated.  They  may  be  considered  as  one  lens  with  a  reciprocal  focal 
distance  equal  to  the  sum  of  the  reciprocal  focal  distances  of  the  component 
lenses ;  e.g.,  the  biconvex  lens  will  gain  in  focal  distance  by  adding  a  positive 
meniscus  (Fig.  5,  a),  while  it  will  lose  when  combined  with  a  plano-concave 
lens  (Fig.  5,  b).  Again,  the  components  may  be  made  of  the  same  sort  of 
glass  or,  what  is  done  much  more  frequently,  two  different  kinds  of  glass  with 
different  refraction  and  dispersion  power  are  selected;  e.g.,  a  combination  is 
made  of  a  plano-concave  lens  of  flint  glass  and  a  biconvex  crown  glass. 

After  what  has  been  said  it  would  seem  that  the  strongest  enlargement 
could  be  obtained  by  the  use  of  a  simple  or  a  combined  positive  lens,  since  it 
would  be  only  necessary  to  use  lenses  with  a  corresponding  short  focal  dis- 
tance. A  biconcave  lens  of  1  mm  focal  distance,  i.e.,  1  mm  curvature  radius, 
should  give  us  an  enlargement  of  250  magnifying  power.  Practically  this  can- 
not be  accomplished,  since  such  small  lenses,  aside  from  the  difficulty  of  their 
manufacture,  are  too  weak,  the  distance  between  object  and  eye  would  become 
too  small,  and  finally  such  lenses  would  possess  the  two  great  shortcomings  of 
every  spherical,  refractive  plane,  namely,  spherical  aberration  and  chromatic 
aberration,  to  such  a  degree  as  to  make  them  useless  in  practice. 

Spherical  Aberration. 

By  spherical  aberration  we  understand  that  peculiarity  of  every 
spherical,  refractive  plane  by  which  only  those  of  the  parallel  rays  of  light  that 


8 

are  nearest  the  optic  axis  meet  in  the  focus,  while  those  nearer  the  periphery 
are  united  nearer  the  lens,  approximating  the  latter  more  and  more  the  nearer 
they  are  to  the  periphery. 

Chromatic  Aberration. 

Since  red  rays  are  refracted  less  than  blue,  the  former  will  have  a  greater 
focal  distance  than  the  latter.  This  phenomenon  has  been  called  chromatic 
aberration. 

To  avoid  spherical  aberration  the  peripheral  rays  are  deflected,  but  also 
can  be  obliterated  by  correct  construction  and  position  of  the  lenses.  For 
instance,  a  biconvex  lens  with  equally  curved  planes  will  show  a  greater  aberra- 
tion than  one  in  which  the  curvature  radii  varies,  bearing  a  fixed  relation  to 
one  another.  A  plano-convex  lens  will  give  less  aberration  than  a  biconvex,  if 
the  piano-surface  of  the  former  is  directed  toward  the  incoming  rays.  To 
avoid  chromatic  aberration  the  optician  combines  lenses  of  different  dispersion 
power.  He  will,  for  instance,  correct  the  dispersion  power  of  the  biconvex 


FIG.  6. 
Keproduction  by  Two  Piano-Convex  Lenses. 

crown-glass  lens  (Fig.  5  fy)  by  a  plano-concave  lens  of  flint  glass,  thereby  sacri- 
ficing, however,  the  refractive  power. 

The  Combined  System. 

In  practical  work  one  never  goes  below  a  focal  distance  of  10  mm  in  a 
single  lens,  hence,  with  such  a  lens,  no  enlargement  greater  than  25  X  is  ever 
obtained.  If  greater  power  is  desired,  two  or  more  lenses,  separated  a  certain 
distance  from  one  another,  are  used.  Such  a  combination  we  find  illustrated  in 
Fig.  6.  Here  we  have  a  plano-convex  lens  (I/1)  of  20  mm  focal  distance. 
The  object  a  b  being  situated  right  in  front  of  the  anterior  focus,  the  lens  pro- 
jects a  reversed  enlarged  real  image  al  b^.  This  image  lying  again  within  the 
focal  distance  of  a  second  plano-convex  lens  (L2)  of  40  mm  focal  distance,  a 
second,  still  more  enlarged,  virtual  and  also  reversed  picture  (referring  to  the 
object)  a2  b2  is  produced.  Hence,  by  projecting  the  image  of  a  primary  posi- 
tive lens  within  the  focal  distance  of  a  second  positive  lens,  we  are  enabled  to 
produce  a  second  enlargement  of  the  image,  and  from  what  has  been  said  it  is 
evident  that  this  enlargement  will  become  greater  as  the  distance  between  the 
two  lenses  is  increased,  i.e.,  dependent  on  how  near  we  bring  the  first  image  to 
the  anterior  focus  of  the  second  lens. 


Course  of  Rays  in  the 
Compound  Microscope. 

In  Fig.  7  things  are  still  more  complicated. 
lenses  into  our  optic  system:  the  plano-convex  L± 


Here  we  have  taken  three 
(20  mm),  the  convex-piano 


L2  (40  mm)  and  the  convex-piano  L3  (30  mm).  The  distance  between  the 
second  and  third  lenses  has  been  so  chosen  as  to  amount  to  half  the  sum  of 
their  focal  distances,  viz.,  35  mm.  The  size  of  the  object  and  its  distance  from 
the  anterior  focus  of  the  first  lens  being  the  same  as  in  the_previously  men- 
tioned case,  the  image  obtained  would  also  be  the  same,  if  lens  L2  had  not  been 
interposed  in  the  course  of  rays,  converging  the  rays  and  producing  the  much 
smaller  image  a.,  b2»  instead  of  a^  b^  This  second  picture  is  projected  within 
the  focal,  distance  of  the  much  more  curved  lens  L3,  which  finally  furnishes  the 
greatly  enlarged  image  a3  bs. 

A  similar  arrangement  we  find  in  our  compound  microscope.  Here, 
too,  an  enlarged  reversed  real  image  of  the  object  is  projected  through  a  lens 
or  rather  a  system  of  lenses,  the  focal  distance  of  which  ranges  between  1.5 
and  40  mm,  since  the  object  is  situated  closely  anterior  to  the  focus  of  this 
system.  The  latter,  being  so  proximal  to  the  object,  is  termed  the  objective 
or  object-glass.  The  lenses  L2  and  L3  are  united  into  a  system,  which,  being 


Course  of  Rays  in  the  Compound  Microscope. 

proximal  to  the  eye  of  the  observer,  has  been  termed  the  ocular  or  eye- 
piece. The  lens  L2  again  is  called  collective  lens,  while  L3  receives  the 
name  eye-lens.  Here,  too,  as  in  the  previous  case,  the  distance  between  the 
eye-lens  and  the  objective  lens  equals  about  half  of  the  sum  of  their  focal  dis- 
tances. On  the  other  hand,  in  the  microscope  the  eyepiece  is  much  more  dis- 
tant from  the  object-glass,  as  in  our  given  example,  the  distance  of  the  upper 
focus  of  the  object-glass  from  the  lower  one  of  the  eyepiece  being  160  mm. 
This  distance  has  been  designated  as  the  optic  or  reduced  tube-length 
(A),  and  is  chosen  so  as  to  project  the  image  produced  by  the  eye-lens  of  the 
eyepiece  at  a  distance  of  250  mm,  i.e.,  in  the  distinct  visual  distance  of  the 
observer. 

Magnifying  Power  of  the 
Compound  Microscope. 

The  enlargement  produced  by  such  a  combined  microscope  is  always 
easily  figured  out  if  the  focal  distances  of  the  object-glass  (F^)  and  the  eye- 
piece (F2)  are  known,  viz.: 

A 


N=          ' 


After  these  initiating  remarks  let  us  proceed  to  the  description  of  a  micro- 
scope, which  seems  especially  adapted  for  our  purpose. 


10 


Description  of  the  Microscope. 

Our  instrument  (Fig.  8)  rests  on  a  horseshoe-shaped  foot  or  base,  sur- 
mounted by  the  barrel  or  body.     Generally  we  find  in  the  latter,  right  over 


eyepiece 


obturator 


nosepiece 


objectives 
opening  in  object-table 


condenser 
handle  of  diaphragm 


screw  for  condenser 


rack  and  pinion 


milled-heads  (coarse  adjustment) 


micrometer-screw 


prism 

object-table  or  stage 
joint 

pillar 


foot 
FIG.    8. 

The  Microscope. 

the  foot,  a  joint,  which  allows  an  oblique  position  of  the  instrument,  thus  per- 
mitting of  a  more  convenient  examination  in  the  sitting  posture.  Closely  above 
this  joint  is  the  square  stage,  intimately  connected  with  the  barrel  and  having 
a  round  opening  in  its  middle.  Here  the  specimen,  fixed  by  clips,  is  deposited 


11 

for  examination.     The  table  divides  the  microscope  into  a  lower  and  upper 

part. 

On  the  inferior  surface  of  the  stage  a  projection  is  found,  the  condenser, 
a  system  of  lenses  set  in  metal,  for  illuminating  purposes.  By  means  of  a 
screw  the  condenser  can  be  adjusted.  By  turning  it  to  4he- right,  you  ap- 
proximate the  apparatus  to  the  stage,  gradually  introducing  it  through  the 
opening  in  the  stage,*  until  the  upper  surface  of  its  superior  lens  is  on  a  level 
with  the  upper  surface  of  the  table.  Turning  to  the  left,  the  condenser  will 
recede  from  the  table,  and  finally  swings  out  to  the  left. 

On  the  lower  portion  of  the  condenser  two  knobs  are  seen,  one  serving  to 
open  and  close  the  diaphragm,  the  second  being  attached  to  a  swinging  ring, 
into  which  colored  (e.g.,  blue)  glass  plates  may  be  inserted,  such  as  are  neces- 
sary, when  working  in  artificial  light. 

Below  the  condenser  the  mirror  will  be  found,  a  silvered  double  mirror, 
having  one  plane  surface  on  one  side  and  a  concave  on  the  other.  It  is  con- 
nected with  the  pillar  by  a  movable  lever  and  suspended  in  a  metal  crescent  so 
as  to  allow  of  any  position. 

Above  the  obje,ct-table  the  pillar  continues  as  a  trigonal  metal  prism, 
visible  only  for  a  few  millimetres,  as  it  is  enveloped  by  a  hollow  metal  cylinder, 
the  barrel  carrier.  The  hollow  of  the  carrier  corresponds  exactly  to  the  size 
of  the  prism,  so  that  the  two  are  in  intimate  relation.  On  top  the  carrier  is 
surmounted  by  a  hoodlike  screw  head,  the  head  of  the  micrometer '-screw, 
in  front  it  is  elongated  into  an  armlike  projection,  which  supports  the  barrel. 

The  barrel  or  tube  is  a  hollow  brass  cylinder.  Above  it  is  mounted 
by  a  ring,  through  which  glides  a  second  graduated  tube,  the  draw-tube. 
The  scale  is  graduated  in  millimetres,  ranging  from  140  to  200  mm  or 
14  to  20  cm,  starting  on  top  with  the  smallest  figure  and  ending  below  with  the 
largest.  These  figures  represent  the  tube  length,  figured  from  the  inferior 
outlet  of  the  barrel,  to  which  the  objective  is  attached,  up  to  the  superior  bor- 
der of  the  draw-tube,  on  which  the  eyepiece  rests.  If  we  draw  out  the  draw- 
tube  we  increase  the  distance  between  objective  and  eyepiece,  and  hence  be- 
tween their  respective  foci.  Thereby  the  magnifying  power  of  the  microscope 
is  increased. 

This  fact  may  be  made  use  of  when  employing  a  weak  objective,  but  is 
objectionable  with  the  stronger  object-glasses,  as  the  latter  have  been  con- 
strued for  a  certain  tube  length,  viz.,  160  mm.  We  must  also  take  into  con- 
sideration that  in  our  instrument  a  nosepiece,  8  mm  thick,  is  inserted  between 
barrel  and  objective,  which  must  be  allowed  for,  so  that  for  all  the  stronger 
enlargements  the  barrel  must  be  drawn  to  152  mm,  i.e.,  the  draw-tube  must 
register  15.2. 

Body  and  arm  are  connected  by  rack  and  pinion,  the  rack  forming  part  of 
the  body  posteriorly,  the  pinion  part  of  the  arm,  the  teeth  engaging  in  recesses 
on  a  transverse  rod,  which  has  screw-heads  on  either  end,  the  coarse  adjust- 
ment. By  turning  these  knobs  the  barrel  is  lowered  or  raised,  thus  approxi- 
mating or  receding  from  the  stage. 

On  the  lower  end  of  the  barrel  the  so-called  nosepiece  is  found,  for  the 
purpose  of  quickly  changing  objectives.  It  consists  of  a  metal  plate  having 
three  arms,  each  arm  being  provided  with  a  threaded  ring,  the  whole  revolving 


12 

on  a  common  axis.  The  plate  itself  has  a  threaded  hole,  by  virtue  of  which  it 
is  attached  to  the  barrel.  When  screwed  in  place  the  three-armed  piece  will 
revolve  around  the  plate  in  such  fashion  as  to  fit  the  rings  exactly  to  the 
lower  barrel  opening.  A  spring  ( X )  in  the  back  of  the  nosepiece  indicates 
whether  the  position  is  accurate,  so  that  one  may  look  right  through  the  barrel 
and  one  ring,  the  other  two  being  covered  by  the  plate. 

The  eyepiece  is  attached  to  the  upper  aperture  of  the  draw-tube,  while 
below,  the  objectives  are  screwed  to  the  rings  of  the  nosepiece.  The  latter 
should  be  placed  in  consecutive*©rder,  viz.,  assuming  objective  3  to  be  in  place, 
first  objective  6  and  then  objective  1/12  should  follow,  when  the  nosepiece  is 
turned  to  the  right. 

Several  of  the  parts  of  the  microscope  spoken  of  require  a  more  thorough 
discussion,  principally  the  objectives,  the  eyepieces,  the  condenser  and 
finally  the  micrometer-screw. 


THE    OBJECTIVE 

The  Significance  of  the 
Objective. 

As  already  explained  in  our  general  discussion  of  refraction  of  rays  in  the 
compound  microscope,  the  purpose  of  the  objective  is  to  furnish  a  real,  re- 
versed, enlarged  image  of  the  object  to  be  examined.  The  latter  simply  being 
enlarged  again  by  the  eyepiece,  it  must  needs  be  free  of  all  refractive  errors, 
free  of  all  spherical  as  well  as  chromatic  aberration.  This  leaves  no  question 
that  the  objective  is  the  most  important  part  of  the  entire  microscope,  being 
of  much  greater  consequence  than  the  eyepiece,  since  we  can  merely  enlarge 
the  objective  image  considerably  by  a  strong  eyepiece,  without  bringing  any 
new  elements  into  the  microscopic  picture.  A  structure  not  previously  re- 
vealed by  the  objective  will  never  be  disclosed  by  an  eyepiece,  no  matter  how 
strong  the  latter  may  be. 

Structure  of  the  Objective. 

In  order  to  comply  with  all  the  requirements,  the  objective  in  the  compound 
microscope  cannot,  as  we  have  supposed  in  our  earlier  discussion,  be  repre- 
sented by  a  single  lens,  but  is  composed  of  a  series  of  parts,  arranged  in  a 
common  metal  setting  in  such  a  fashion  that  all  are  exactly  concentric,  i.e.,  all 
lie  in  the  same  optic  axis. 

As  a  rule,  the  different  members  are  connected  to  one  another  at  a  fixed 
distance,  although  in  rare  instances  the  distance  may  be  altered  within  certain 
limits. 

Each  member  is  attached  to  the  lower  end  of  a  brass  tubule,  and  these 
tubules,  sometimes  separated  by  an  interposed  metal  tube,  are  screwed  one 
into  the  other.  According  to  the  number  of  lenses  we  have  double,  triple  and 
quadruple  systems.  Commonly  each  successive  member  is  larger  than  the  pre- 
ceding, making  the  first  member,  the  so-called  anterior  or  front  lens,  the 
smallest  of  all. 

This  front  lens  is  the  principal  or  sole  agent  in  magnifying,  while  the  re- 
maining lenses  or  correction  lenses  have  as  their  object  the  clearing  out  of 
errors  from  the  image  furnished  by  the  front  lens.  Hence  the  size  of  the  front 


13 

lens  will  give  us  an  estimate  of  the  power  of  the  entire  objective,  so  that  we 
are  able  to  say  that,  the  larger  the  front  lens  is,  the  lower  will  be  the  power  of 
the  objective.  Since  low-power  objectives  have  a  greater  focal  distance  that 
high  power,  the  object,  however,  necessarily  being  without  the  focal  distance 
of  the  objective,  it  will  be  seen  that  during  a  sharp  focus  the  distance  between 
object  and  front  lens  of  objective,  the  so-called  free  object  distance ,  is 
greater  in  low-power  objectives  than  in  high.  For  example,  with  an  objective 
of  42  mm  focal  distance  we  will  get  an  object  distance  of  40  mm,  while  one  of 
1.8  mm  will  have  only  0.17.  In  the  latter,  therefore,  the  front  lens  must  be 
brought  very  near  to  the  object. 

In  low-power  objectives  this  front  lens  is  mostly  composed  of  several  single 
lenses;  in  high-power  object-glasses,  however,  there  is  generally  one  single 
plano-convex  (hemispherical)  lens,  the  plane  surface  being  turned  toward  the 
object.  Most  of  the  weaker  objectives  are  doublets,  one  combination  lens  fol- 
lowing the  combination  front  lens.  Here  both  members  take  part  in  the  mag- 
nifying as  well  as  correcting  process.  Medium  objectives  have  two  combined 
correction  lenses  above  the  front  lens,  the  latter  furnishing  the  power,  while 
in  the  most  powerful  object-glasses  we  find  between  front  lens  and  the  correc- 
tion systems  a  fourth  simple  lens,  either  plano-convex  or  a  positive  meniscus. 

Achromatic  and  Apochromatic 
Objectives. 

The  individual  systems,  and  the  lenses  composing  them,  are  made  of  dif- 
ferent artificial  glass  compounds,  each  possessing  its  own  refractive  and  dis- 
persive qualities.  Recently  objectives  have  been  constructed  with  lenses  made 
partly  of  a  single  mineral,  namely  fluor  spar.  As  regards  refractive  index, 
this  mineral  only  equals  crown  glass,  but  it  excels  all  artificial  glasses  in  its 
dispersive  power.  By  the  use  of  fluor  spar  the  greatest  color  purity  of  the 
microscopic  image  has  been  obtained.  Objectives  made  of  glass  solely  are 
known  as  achromatic,  those  in  which  fluor  spar  is  used  as  apochromatic. 
For  our  purpose  the  latter  may  be  disregarded,  being  much  more  expensive 
than  the  former  on  account  of  the  high-priced  raw  material,  without  yielding 
much  better  results  for  our  work. 

Oil  Immersion  Objectives. 

Finally,  there  is  one  more  very  important  point  to  deal  with.  Heretofore 
we  have  always  assumed  that  the  rays  emanating  from  the  object  enter  the 
front  lens  directly  through  the  air.  In  reality  this  is  not  so,  as  in  most  cases 
our  object  is  covered  with  a  thin  plate  of  glass,  the  cover-glass.  The  rays  of 
light  therefore  are  first  refracted  by  this  cover-glass  before  reaching  the  front 
lens  or,  to  be  more  exact,  they  emanate  from  the  object,  pass  through  the 
cover-glass,  thence  into  the  air  and  finally  enter  the  front  lens.  The  sealing 
medium  possessing  about  the  same  refractive  index  as  the  cover-glass,  the 
rays  of  light  emerging  from  the  object  will  pass  the  cover-glass  unrefracted, 
but  become  refracted  at  the  surface  of  the  cover-glass,  when  the  following 
takes  place :  since  they  pass  from  glass  to  air,  they  are  everted  from  the  optic 
axis,  so  that  the  angle  of  exit  is  greater  than  the  entrance  angle,  hence  a  much 
smaller  part  of  the  light-rays  enters  the  objective  than  we  have  heretofore 
supposed.  This  fact  is  not  of  great  importance  when  dealing  with  objectives 


having  a  large  front  lens,  i.e.,  low-power  objective,  but  is  very  noticeable  when 
a  small  front  lens  is  used.  This  condition  is  illustrated  in  the  right  half  of 
Fig.  9,  while  the  left  half  shows  the  remedy  for  it.  All  we  have  to  do  is  to 
interpose  between  front  lens  and  cover-glass  a  medium  of  the  same  refractive 
index  as  the  latter,  and  the  ray  a  b,  otherwise  refracted,  will  now  reach  the 
objective  unrefracted. 

The  interposing  of  such  a  medium,  which  necessarily  must  be  liquid,  is 
done  without  difficulty,  since  the  high-power  objectives  have  but  a  small  object 
distance.  A  drop  of  condensed  cedarwood  oil  is  placed  on  the  cover-glass,  the 
barrel  is  lowered  so  far  as  to  allow  the  front  lens  to  dip  into  the  drop  and  thus 
a  homogeneous  connection  between  object  and  objective  is  effected.  Such  ob- 
jectives are  called  homogeneous  immersion  objectives  or  oil-immer- 
sion lenses  f  and  are  to  be  differentiated  from  immersion  objectives,  where 


front  len 


FIG.  9. 
The  Effect  of  Immersion. 


no  oil  but  some  other  liquid  is  used  as  a  medium.  We  thus  hear  of  immersion 
systems,  where  water  is  used,  viz.,  a  liquid  of  lower  refractive  index  than  oil, 
or  those  in  which  monobrome  naphthalin  is  the  medium,  the  latter  of  a 
higher  refractive  power,  which  is  used  when  a  front  lens  of  strongly  refractive 
flint  glass  is  engaged. 

Angular  Aperture 

From  previous  remarks  it  will  be  seen  that  an  immersion  lens  is  superior 
to  a  dry  lens  of  the  same  focal  distance  and  aperture,  since  in  the  latter  the 
available  aperture  of  the  objective  is  not  fully  utilized.  The  angular  aper- 
ture ,  or,  in  short,  the  angle  of  an  objective,  are  terms  used  to  designate  the 
angle,  having  its  apex  at  the  central  point  of  the  object,  its  arms  extending  to 
the  periphery  of  the  front  lens  of  the  objective,  when  the  object  is  in  sharp 
focus.  Its  size  is  dependent  upon  the  free  object  distance  of  the  objective  and 
the  diameter  of  the  front  lens.  The  medium  separating  objective  and  cover- 
glass  must  also  be  taken  into  consideration  when  calculating  the  angle.  The 
figure  obtained  when  considering  both,  is  the  numerical  aperture  found  by 


15 

multiplying  the  refractive  index  of  the  interposed  medium  with  the  sinus  of 
half  of  the  angular  aperture : 

a=n  .  sin  u. 

Since  in  the  dry  system  n  equals  1,  the  aperture  in  such  4s  equal  to  half 
the  opening  angle.  With  water  immersion  this  value  must  be  multiplied  with 
1.33,  with  oil  immersion  by  1.51,  and  with  monobromc  naphthalin  by  1.62.  As 
the  angle  u  can  never  attain  90°,  its  sinus,  and  hence  the  aperture  of  every  dry 
system,  is  always  less  than  1.  On  the  other  hand  a  may  be  larger  than  1  when 
immersion  systems  are  used. 

The  Requisites  of 
an  Objective. 

A  good  objective  should  possess  the  following  qualities:  1.  It  must  be 
achromatic,  i.e.,  red  and  blue  rays  should  unite  absolutely.  2.  A  planatic 
image  should  be  obtained.  3.  All  rays  emanating  from  one  point  of  the 
object  should,  as  far  as  possible,  be  united  in  one  point  of  the  image,  i.e., 
sharp  pictures  should  be  furnished;  no  blurring  should  take  place.  4.  It 
should  furnish  detail,  i.e.,Whe  finest  structures  of  our  specimen  must  be  re- 
produced. 

The  achromatic  correction  of  the  objective,  as  we  have  seen,  is  brought 
about  by  employing  lenses  of  varying  dispersion  properties.  To  procure 
aplanasia  and  the  greatest  sharpness,  spherical  aberration  must  be,  as  far  as 
possible,  obliterated.  As  has  been  previously  mentioned,  the  different  mem- 
bers of  the  objective  are  made  up  of  lenses  of  various  refractive  powers  and 
thus  the  preceding  member  is  corrected  by  the  succeeding  one.  The  optician 
at  times  even  goes  as  far  as  to  overcorrect  such  a  member,  subsequently  bal- 
ancing this  overcorrection  by  the.  following  lens.  The  detailing  power  finally 
is  wholly  dependent  upon  the  aperture  and  is  in  direct  proportion  with  the 
increase  of  the  angle. 

Objectives  are  marked  by  their  manufacturers  either  with  figures  (Arabic 
or  Roman)  or  with  letters,  giving  a  higher  number  or  a  later  letter  of  the 
alphabet  to  the  stronger  objective,  and  vice  versa.  Immersion  objectives,  on 
the  other  hand,  and  also  the  briefly  mentioned  apochromatics,  are  known  by 
their  focal  distance,  the  former  expressed  in  inches,  the  latter  in  millimetres. 
Objective  1/12  therefore  will  possess  a  focal  distance  of  1/12  inch,  i.e., 
2.1  mm. 

Objective  3. 

After  these  general  preliminaries  let  us  consider  more  closely  those  ob- 
jectives which  are  of  special  interest  to  us  (Figs.  10-12).  Turning  to  figure 
10  we  find  a  section  through  objective  3.  Here  we  have  the  type  of  a  weak 
objective,  consisting  of  only  two  lenses,  these  two  members,  however,  being 
double  lenses.  The  first  member  has  a  diameter  of  6  mm  and  consists  of  a 
plano-concave  flint-glass  lens  and  a  biconvex  crown-glass  lens,  the  focal  dis- 
tance being  15  mm.  The  second  member,  which  is  of  the  same  combination, 
has  a  diameter  and  curvature  radius  3  mm  greater  than  the  former,  the  focal 
distance  being  29  mm  with  a  distance  from  the  first  member  of  14  mm.  Thus 
the  entire  objective  has  a  focal  distance  of  16.2  mm,  a  free  object  distance  of 


16 


5.5  mm  and  a  numerical  aperture  of  0.3.  The  objective  alone  furnishes  an 
enlargement  of  10.3,  with  the  weakest  eyepiece,  however,  one  of  41 ;  in  the 
latter  case  it  still  covers  a  field  of  2.1  mm  diameter. 

Objective  6. 

Fig.  11  (objective  6)  represents  a  system  of  medium  strength.  The  three 
members  composing  it  are  placed  in  separate  metal  shells  at  distances  0.15, 
respectively  0.65  mm.  The  second  shell  is  screwed  over  the  third,  while  the  first 
envelops  both  the  former,  being  screwed  to  the  base  of  the  third,  the  upper 
extremity  of  which  is  in  direct  contact  with  the  body  proper  of  the  objective. 
The  first  division  consists  of  a  hemispherical  crown-glass  lens  of  3.5  mm  focal 
distance  and  about  the  same  diameter;  the  two  succeeding  members  are  made 
up  each  of  a  plano-concave  flint-glass  and  a  biconvex  crown-glass  lens.  The 
focal  distance  of  12.5  mm  in  the  second  is  raised  to  17.6  mm  in  the  third 
member,  while  the  diameters  are  6  and  7  mm,  respectively,  bringing  the  total 


FIG.  10. 

Objective  3 
(Leitz). 


FIG.  11. 

Objective  6 
(Leitz). 


FIG.  12. 

Objective  1/12, 

Homogeneous  Immersion 

(Leitz). 


focal  distance  down  to  4.0  mm.  The  magnifying  power  of  the  objective  itself 
is  48,  with  a  free  object  distance  of  only  0.42  mm,  while  its  angular  aperture 
has  increased  to  0.82.  With  the  weakest  eyepiece  one  may  obtain  an  enlarge- 
ment of  192  with  this  object-glass,  i.e.,  it  is  five  times  more  powerful  than 
objective  3,  but  the  diameter  of  the  visual  field  here  only  amounts  to  0.48  mm, 
i.e.,  one- fourth  of  the  objective  3. 

Objective  1/12. 

Fig.  12  acquaints  us  with  a  powerful  objective,  an  immersion  system.  It 
is  composed  of  four  members,  the  first  two  being  single,  the  last  two  double 
lenses.  The  distances  are  very  minute:  0.02,  0.1,  0.15  mm  respectively.  Each 
division  has  its  own  shell ;  the  setting  of  the  first  is  made  of  aluminum.  The 
various  shells  are  attached  to  one  another  in  such  fashion  that  1  is  screwed 
to  2,  2  to  4,  3  to  4,  and  4  to  the  body  proper  of  the  objective.  The  first  mem- 
ber consists  of  a  single  hemisphere,  made  of  crown  glass  with  a  diameter  of 
1.96  mm  and  a  focal  distance  of  1.55  mm.  The  second  is  a  positive  meniscus 
with  a  focal  distance  of  4.92  mm,  while  the  third  and  fourth  are  again  piano- 


17 

convex  flint-crown-glass  double  lenses  of  11.14  and  29.28  mm  focal  distance 
respectively.  The  entire  object-glass  possesses  a  focal  distance  of  1.85  mm, 
a  free  object  distance  of  only  0.17  mm  and  an  aperture  of  1.30.  It  has  per 
se  a  magnifying  power  of  105,  which  increases  to  420  with  the  lowest  eyepiece, 
and  a  visual  field  of  0.24  mm  is  covered  with  this  objective.  — 

Objectives  for  Very  Low 
Variable  Enlargement. 

Often  a  want  is  felt  for  a  lower  system  with  a  correspondingly  larger  visual 
field.  Objective  la  is  most  adapted  for  that  purpose.  Since  this  type  is  new 
to  us,  we  will  give  it  brief  mention  here.  It  consists  of  two  achromatic  double 
lenses  in  a  movable  setting,  so  that  they  may  be  approximated  or  withdrawn 
from  one  another  by  means  of  a  screw  to  the  extent  of  about  5  mm.  The  front 
member  consists  of  a  biconcave  flint-glass  lens  in  back  of  a  plano-convex 
crown-glass  lens,  and  has  a  negative  common  focal  distance  of  14.8  mm.  The 
upper  member  is  made  up  of  a  plano-convex  flint-crown-glass  double  lens  of 
24.0  mm  focal  distance.  When  both  systems  are  at  a  maximum  distance  (30 
mm),  the  common  focal  distance  is  24  mm.  When  approximated  as  far  as 
possible,  this  increases  to  33  mm.  Here  the  magnifying  power  proper  falls 
from  3.1  to  2.0  and  the  object  distance  is  thus  increased  from  2.0  to  14.0  mm, 
while  the  numerical  aperture  decreases  from  0.07  to  0.05.  With  the  weakest 
eyepiece  a  power  of  8 — 12  is  obtained  with  a  visual  field  of  about  10  mm  diame- 
ter. The  distance  of  the  two  lenses  from  each  other  can  be  read  on  a  scale 
marked  externally. 

THE    EYEPIECE— (Ocular) 

The  eyepiece  consists  of  a  metal  cylinder  fitting  snugly  into  the  draw-tube, 
the  component  lenses  being  mounted  on  either  side.     On  its  upper  extremity 
we  find  a  metal  ring  protruding,  preventing  the  ocular 
from  sliding  through  the  tube. 

The  Huyghen  Eyepiece. 

There  are  various  sorts  of  oculars,  principally  the 
Huyghen,  the  Ramsden,  and  the  so-called  compen- 
sation eyepiece.  We  will  only  consider  the  first.  The 
Huyghen  ocular  (Fig.  13)  is  composed  of  two  plano-con- 
vex lenses,  one  of  which,  the  eye-lens,  is  placed  at  the 
upper,  the  other  the  collective  lens,  on  the  lower  end, 
the  convexity  of  both  being  toward  the  objective.  Be- 
tween the  two  lenses  we  find  a  diaphragm  on  the  in- 
terior of  the  ocular.  FIG.  13. 

The  eye-lens  is  always  the  stronger,  having  only  half  Huyghen's  Eyepiece 
the  focal  distance  of  the  collective  lens.  As  previously  (Leitz). 

mentioned,   the   distance   between   the   two   lenses   is   half 

of  the  sum  of  their  focal  distances.  Hence  these  focal  distances,  and 
thus  the  total  focal  distance  of  the  ocular,  may  easily  be  determined  ,by 
measuring  the  distance,  i.e.,  the  length  of  the  eyepiece.  For  example,  the  dis- 


18 

tance  (e)  of  the  two  lenses  in  our  eyepiece  ///  measures  36  mm  in  round 
figures;  using  the  equation:  e=  — — —  and  granting  that  f2=2f^  f]?  i.e., 

m 

the  focal  distance  of  the  eye-lens,  must  be  24  mm ;  /2,  i.e.,  that  of  the  collective 
lens,  will  be  48  mm,  and  the  total  focal  distance  of  the  ocular,  /,  according 

f  '  f 

to  the  formula  /=  — — —  will  be  found  to  be  32  mm.     The  lower  focus  of  the 
e 

ocular  lies  between  its  lenses,  the  upper  externally  above  the  eye-lens.  It  is 
of  great  importance,  as  far  as  the  Huyghen  ocular  is  concerned,  that  it  is  con- 

f  +  f 

structed  in  accordance  with  formula  e=    l  &        a»  nearly  as  possible,  for  it  is 

& 

due  to  that  fact  only,  that,  in  spite  of  the  eye-lens  as  well  as  the  collective  lens 
being  simple  non-achromatic,  the  image  furnished  by  the  objective  is  enlarged 
without  aberration. 

Significance  of  Eyepiece. 

As  we  have  previously  observed,  the  image  produced  by  the  objective  is 
first  reduced  by  the  collective  lens,  the  latter  collecting  the  rays  coming  from 
the  former.  The  diaphragm  is  found  in  the  plane,  where  this  reduced  real 
image  is  produced,  which  latter  may  be  demonstrated  as  follows :  Focus  some 
object,  and  after  removing  the  eye-lens  place  a  properly  cut  piece  of  thin 
tissue-paper  on  the  diaphragmatic  opening;  if  the  collective  lens  is  now  also 
removed,  a  much  enlarged  image  will  be  projected  on  the  diaphragm,  which 
can  be  focused  sharply  by  adjusting  the  draw- tube. 

Thus  our  real  image  is  simply  enlarged  by  the  eye-lens  and  brought  into 
exact  visual  distance  of  the  observer.  At  the  same  time,  of  course,  the  rim  of 
the  diaphragm  is  projected,  so  that  our  field  is  now  bordered  by  a  sharply 
defined  periphery. 

Since  this  enlargement  distributes  the  same  amount  of  light  over  a  much 
greater  space,  the  picture  thus  produced  will  be  short  of  light,  and  this  loss 
naturally  will  increase  with  the  strength  of  the  eyepiece.  Hence  a  certain  limit 
is  set  to  ocular  magnifying,  and  a  power  greater  than  twelve  should  not 
be  used,  at  least  not  with  a  Huyghen  eyepiece. 

Eyepieces  are  generally  numbered  in  Roman  or  Arabic  figures,  and  naturally 
a  corresponding  decrease  in  size  of  the  lenses  and  in  length  of  the  eyepiece  is 
noted  in  proportion  with  the  decrease  in  strength. 


ILLUMINATION 

Illumination  by  Mirror. 

So  far  we  have  assumed  that  our  object  itself  sends  out  rays  of  light  spon- 
taneously, which  in  reality  is  not  the  case.  We  therefore  illuminate  our  speci- 
men artificially  in  order  to  enable  it  to  radiate  light.  This  illumination  may 
be  brought  about  in  two  ways:  either  from  above  when  the  section  is  imper- 
meable to  light,  or  from  below  through  the  opening  in  the  stage.  The  former 
method,  using  reflected  light,1  is  of  little  interest  to  us,  as  most  all  examina- 

1  In  this  method  the  Bull's  eye  condenser,  so-called,  is  used. — (The  Translator.) 


19 

tions  are  made  with  the  aid  of  the  latter,  the  transmitted  light,  method. 
Illumination  here  takes  place  from  below  by  means  of  the  movable  mirror, 
which,  as  stated  before,  is  plane  on  one  side,  concave  on  the  other. 

Assuming  that  we  are  using  the  plane  mirror,  a  luminous  cloud  furnishing 
the  source  of  light,  the  parallel  rays  emanating  from  the  latter  are  caught  by 
our  mirror  and  reflected  into  the  specimen.  Each  point  of  the  latter  thus 
sends  its  slightly  diverging  bundle  of  rays  into  the  objective.  As  each  bundle 
must  needs  completely  fill  out  the  angular  aperture  of  the  objective,  in  order 
to  obtain  good  illumination,  we  can  use  this  plane  mirror  only  with  objectives 
of  a  small  aperture,  to  wit,  weak  objectives.  When  using  stronger  objectives, 
we  must  have  bundles  of  light,  which  have  a  greater  divergence,  and  these  will 
only  be  furnished  by  a  concave  mirror,  which  causes  the  parallel  rays  to  con- 
verge and  which  is  so  adjusted  as  to  focus  in  the  objective.  Rays  of  about  40° 
divergence  can  be  obtained  from  this  mirror,  which  is  absolutely  sufficient  for 
medium  strength  objectives. 

Illuminating  Apparatus. 
Condensers. 

For  high  power  enlargements  still  more  divergent  bundles  will  be  required, 
in  order  to  exhaust  the  high  apertures  of  the  ob- 
jective, and  it  is  in  these  cases  that  an  extra  sys- 
tem of  lenses  must  be  interposed  between  mirror 
and  specimen,  commonly  known  as  the  Abbe 
condenser. 

This  illuminating  apparatus  is  composed  of 
two  lenses  (Fig.  14),  which  are  snugly  inserted 
in  a  metal  case.  The  entire  system  can  be  ad- 
justed higher  or  lower  and  even  everted  entirely 

by  a  screw,  seen  in  Fig.  8.     The  upper  of  the  two  Abbe's  Condenser, 

lenses  is  more  than  a  hemisphere,  while  the  lower 

one  is  a  biconvex  lens.  The  two  lenses  combine  to  form  a  strong  collecting  sys- 
tem, rendering  the  rays,  coming  from  the  plane  mirror,  strongly  convergent, 
so  that  they  emerge  strongly  divergent.  Our  condenser  has  an  aperture  of 
1.20,  furnishing  bundles  of  rays  of  104°  divergence.  The  minute  loss  of  lumi- 
nosity, caused  by  the  air  between  condenser  and  slide,  may  be  obviated  by  a 
drop  of  immersion  oil  so  applied  as  to  fill  out  this  air-space. 

Since,  in  most  cases,  the  entire  amount  of  light  furnished  by  mirror  or  con- 
denser is  not  required  nor  even  desirable,  the  cone  of  light  is  regulated  by  an 
adjustable  device,  named  the  diaphragm,  which  is  interposed  between  mirror 
and  condenser  and  is  capable  of  narrowing  the  cone  of  light  in  its  course  from 
the  mirror  to  the  condenser  to  a  minimum. 

The  Iris  Diaphragm. 

We  utilize  for  this  purpose  the  so-called  Iris  diaphragm,  which  is  found 
closely  below  the  lenses  of  the  condenser,  being  attached  to  the  metal  case  con- 
taining the  latter.  Such  an  Iris  diaphragm  consists  of  a  number  (12 — 18)  of 
thin  small  metal  plates,  each  resembling  a  sickle  or  crescent  with  a  short  handle, 
the  point  being  obliquely  cut  off.  These  are  arranged  in  a  thin  metal  drum  in 
such  a  manner,  that  they  cover  one  another  almost  entirely  when  the  diaphragm 


20 

is  open  (Fig.  15  a),  leaving  only  their  ends  visible,  thus  forming  a  broad  ring. 
Each  crescent  forms  a  lever,  its  fixed  point  being  located  in  the  handle.  A 
small  rod  is  found  on  the  distal  end  of  each  crescent,  which  plays  in  a  cor- 
responding minute  moat.  These  depressions  are  arranged  in  a  radiating  man- 
ner on  a  metal  ring,  which  is  provided  with  a  lever,  the  diaphragm  lever 
(Figs.  8  and  16  a).  If  the  lever  be  turned  to  the  right,  all  the  crescents  ad- 
vance evenly  toward  the  middle,  thus  bounding  a  polygon,  having  as  many 


FIG.  15. 

Iris  Diaphragm.     The  Cap  and  Lever  have  been 
Removed,    a,  Completely  Open;  &,  Closed. 

sides  as  there  are  crescents  and  being  nearly  circular  (Fig.  15  6).     The  more 
the  lever  is  turned  to  the  right,  the  smaller  will  be  the  diaphragmatic  opening, 


FIG.  16. 
Iris  Diaphragm,    a,  Lever-Ring;  1),  Cap. 

the  minimum  being  one  millimetre  diameter.     This  well-devised  apparatus  thus 
enables  us  to  regulate  the  light  ad  libitum. 

Under  certain  circumstances,  especially  when  using  low-power  lenses,  it  is 
of  advantage  to  be  able  to  switch  out  the  condenser  entirely.  The  place  of 
the  diaphragm  will  then  be  taken  by  the  so-called  cylinder  diaphragm, 
which  can  be  fitted  into  the  opening  in  the  stage  and  which  is  manufactured 
with  variously  sized  openings.  The  light  can  thus  be  regulated  by  the  various 
sizes  of  cylinder  diaphragm  used,  but  aside  from  this  the  amount  of  illumina- 
tion can  also  be  reduced  by  lowering  the  same  cylinder  to  a  desirable  level. 

Source  of  Light. 

The  best  source  of  light  for  our  microscope  is  a  light  cloud,  a  blue  sky 
being  unfavorable,  while  direct  sunlight  must  be  absolutely  avoided.  Of  the 


artificial  lights  the  most  serviceable  and  convenient  is  the  gas-mantle  burner. 
It  is  a  good  plan  to  place  a  reflector  behind  the  lamp  and  to  interpose  a  sheet 
of  tissue-paper  on  a  wooden  frame  between  lamp  and  mirror,  at  least  when  low 
power  is  used. 

THE    MICROMETER-SCREW 

Micrometer-Screw. 

Lastly,  it  behooves  us  to  speak  of  the  micrometer-screw,  which  constitutes 
by  far  the  most  important  mechanical  part  of  the  microscope  and  requires 
the  most  careful  manipulation.  Looking  at  Fig.  17  we  will  easily  understand 
its  construction.  Here  we  find  the  upper  end 

of  the  pillar,   the  hoodlike   covering   of  the  ^^X 

micrometer-screw  having  been  removed.  As 
previously  mentioned,  the  pillar  above  the 
stage  takes  on  a  prismatic  shape  and  is  en- 
closed by  the  carrier.  This  prism  ends  in  a 
cylindro-conical  knob,  projecting  from  the 
carrier,  as  shown  in  our  figure.  This  plug 
as  well  as  the  prism  are  hollow,  the  former 
being  slit  in  addition  oh  its  top.  Through 
this  slit  a  bridge  is  inserted,  which  is  screwed 
to  the  top  of  the  carrier.  A  strong  spiral 
spring,  lodged  in  the  hollow  of  prism  and 
plug,  exerts  pressure  against  this  bridge, 
thus  pressing  the  bridge  and  the  intimately 
connected  carrier  upward.  This  pressure 
is  counteracted  by  the  micrometer-screw 
proper,  a  short,  strong  steel  screw  of  per- 
fect execution,  its  freely  movable  tip  pressing 
against  a  depression  in  the  bridge.  Turning 

the  screw  to  the  right  we  will  compress  the  spring  and  thus  lower  the  tube- 
carrier.  In  this  manner  the  carrier  may  be  lowered  until  its  lower  end  ap- 
proximates the  enlargement  of  the  pillar  found  just  above  the  stage  (Fig.  8), 
in  other  words,  until  the  entire  prism  is  covered.  Turning  to  the  left,  the  car- 
rier, pushed  upward  by  the  spring,  is  raised ;  this  can  be  done  until  the  bridge 
touches  the  ceiling  of  the  slit,  after  which  the  carrier  will  cease  to  rise,  and  if 
the  turning  is  continued,  the  micrometer-screw  will  simply  be  taken  out. 

We  may  add  that  the  different  makes  of  micrometer-screws  vary  essentially 
in  their  construction. 


FIG.  17. 
Micrometer-Screw. 


SOME  RULES  FOR  THE  USE  OF  THE  MICROSCOPE 

The  Procuring  of 
a  Microscope. 

In  the  manufacture  of  microscopes  Germany  has  taken  first  place  for  years 
past,  and  such  renowned  firms  as  Zeiss,  in  Jena,  Leitz  and  Seibert,  in  Wetzlar, 
Winkel,  in  Goettingen,  Voigtlander,  in  Brunswick,  and  Reichert,  in  Vienna, 


22 

furnish  excellent  instruments,  which  can  be  said  to  be  equally  efficient  in  their 
essentials.  It  is  therefore  partly  a  matter  of  personal  preference  or,  perhaps, 
partly  a  pecuniary  question,  which  make  should  be  selected  by  the  prospective 
buyer. 

An  instrument  answering  all  the  requirements  of  a  beginner  may  be  bought 
nowadays  at  a  price  of  300  to  400  marks  (75  to  100  dollars),  and  such  an  in- 
strument will,  moreover,  prove  satisfactory  for  the  work  of  the  general  prac- 
titioner. 

The  objectives  should  be  thoroughly  tested  as  to  their  efficiency,  and  their 
selection  should  best  be  left  to  an  expert.  One  should  also  convince  himself  of 
the  faultless  construction  of  the  micrometer-screw  by  sharply  focusing  at 
different  heights. 

The  Care  of  the  Microscope. 

It  scarcely  needs  mention  that  an  instrument  of  such  minute  precision  as 
the  microscope  requires  the  most  scrupulous  care  and  delicate  manipulation. 
It  should  be  protected  from  dust  by  replacing  it  in  the  case  after  use,  or,  still 
better,  by  keeping  it  under  a  bell-glass  jar.  In  transporting  the  microscope 
one  should  make  it  a  rule  to  always  grasp  the  foot  and.  not  the  tube-carrier,  as 
is  generally  practised. 

The  Cleaning  of  the  Objective. 

The  objectives  require  the  most  rigid  care.  If  the  front  lens  is  soiled,  the 
entire  objective  should  be  unscrewed  from  its  nosepiece  and  the  lens  cleaned 
with  a  very  soft  cloth,  preferably  a  well-laundered  handkerchief.  We  hesitate 
to  recommend  the  chamois  leather,  which,  although  very  useful,  is  soon  soiled. 
If  the  lens  cannot  be  cleaned  satisfactorily  with  the  dry  cloth,  the  latter  should 
be  moistened  with  alcohol.  If  the  front  lens  is  clean  and  still  the  objective  does 
not  furnish  a  clear  picture,  it  will  be  best  to  have  the  optician  look  it  over. 
Under  no  circumstances  should  the  beginner  attempt  to  disarticulate  the  com- 
ponent lenses  with  a  view  of  cleaning  the  same  separately.  It  goes  without 
saying,  that  the  objective,  or  more  particularly  the  front  lens,  must  be  pro- 
tected from  all  acids,  alkalies  or  such  solutions  as  are  solvents  of  fats  and 
resins. 

The  microscope  should  never  remain  without  an  eyepiece  or  without  proper 
apposition  of  the  nosepiece,  as  otherwise  dust  will  accumulate  on  the  interior, 
making  it  difficult  to  keep  the  lenses  clean. 

The  Focussing  of  a  Field. 

It  seems  advisable  to  say  a  few  words  regarding  the  focussing  of  a  slide,  a 
process  which  often  proves  quite  difficult  for  the  beginner.  We  found  that  each 
objective  has  its  own  fixed  object  .distance,  and  that  the  latter  decreases  with 
the  increasing  strength  of  the  lens.  When  handling  a  high-power  lens  it  may 
thus  easily  happen  that  the  front  lens  comes  in  too  intimate  relation  with  the 
cover-glass.  One  of  two  things,  or  both,  may  happen.  Either  the  cover-glass 
will  be  crushed  and  the  specimen  ruined  or,  what  is  more  serious,  the  front  lens 
may  be  loosened  from  its  setting.  This  might  especially  happen  when  using 
an  immersion  lens,  where,  as  we  have  found,  the  metal  setting  of  the  minute 
hemisphere  lies  attached  only  to  a  very  small  ring. 


23 

It  is  an  excellent  plan  for  the  beginner  to  establish  the  rule  to  examine 
first  with  a  low-power  lens.  Any  particular  part  of  the  field  should  then  be 
moved  to  the  middle.  It  will  then  only  be  necessary  to  shift  the  nosepiece  and 
to  use  the  fine  adjustment,  since  the  length  of  the  objectives  has  been  graduated 
in  a  manner  so  as  to  make  the  coarse  adjustment  for  all  objectives  the  same. 

Low-power  objectives,  e.g.,  as  No.  3,  have  such  a  great  distance  as  to 
render  the  focussing  safe.  If  trouble  should  be  encountered  here  also,  it  is 
best  to  proceed  by  raising  the  barrel  rather  than  lowering  it.  The  front  lens 
is  first  brought  down  close  to  the  cover-glass,  then,  while  looking  in  the  micro- 
scope, the  barrel  is  raised  by  means  of  the  coarse  adjustment  screw,  until  the 
picture  appears. 

When  the  oil-immersion  lens  is  to  be  used,  that  portion  of  the  specimen  is 
brought  to  the  middle  of  the  field,  focussing  first  with  a  low-power  and  then 
with  a  medium  lens,  the  barrel  is  then  raised  and  the  nosepiece  turned.  A 
small  drop  of  immersion  oil  is  placed  in  the  middle  of  the  cover-glass  and  the 
barrel  lowered  until  the  front  lens  comes  in  contact  with  the  drop  of  oil.  If 
a  small  drop  of  oil  has  been  used,  the  image  will  now  be  visible,  though  indis- 
tinct, and  all  that  remains  necessary  is  the  use  of  the  micrometer-screw. 
When  the  examination  has  been  completed,  the  front  lens  and  cover-glass  are 
cleansed  with  a  cloth  moistened  with  concentrated  alcohol. 

During  the  entire  examination  the  hand  should  be  kept  on  the  micrometer- 
screw,  the  latter  following  each  glance  of  the  observer  and  making  it  possible 
for  the  eye  to  penetrate  the  various  depths  of  the  specimen ;  hence  its  great  im- 
portance to  our  instrument.  The  micrometer-screw  should  never  be  used  for 
coarse  adjustment,  and  one  should  always  observe  that  the  prism,  mentioned 
before,  is  visible  at  all  times.  If  any  resistance  is  felt  when  focussing,  the 
cause  of  same  should  be  found  before  any  further  adjusting  is  attempted.  The 
micrometer-screw  will  be  found  lowered  to  its  limit  or  the  front  lens  has  touched 
the  cover-glass.  If  the  turning  of  the  micrometer-screw  has  no  effect  on  the 
focus,  it  wil1  be  found  to  have  passed  its  upper  limit.  Too  great  a  part  of  the 
prism  will  be  visible  in  this  case,  and  the  fine  adjustment  must  be  lowered  until 
the  correct  distance  is  attained. 


General    Microtechnique 


Definition  and  Purport 
of  Microtechnique. 

By  microtechnique  we  understand  the  totality  of  all  those  methods  which 
serve  to  produce  a  microscopic  slide.  This  technique  depends  on  the  one  hand 
on  the  peculiarities  of  our  microscope,  on  the  other  it  is  governed  by  the  prop- 
erties of  the  specimen  about  to  be  examined.  We  will  best  make  ourselves  clear 
by  citing  a  simple  practical  example. 

Let  us  suppose  that  we  are  to  prepare  a  specimen  of  the  human  kidney. 
First  we  are  confronted  with  the  proposition  of  making  a  section.  This 
section  must  not  be  made  in  a  coarse,  superficial  manner,  but  must  be  so 
perfected  that  a  transparent  preparation  is  obtained,  since  we  want  to  ex- 
amine our  slide  with  transmitted  illumination.  We  can  either  make  thin  sec- 
tions with  the  cutting  instruments  designed  for  this  purpose,  or  we  may  sepa- 
rate minute  particles  by  means  of  scissors,  and  then  still  reduce  their  size  by 
teasing  with  teasing  needles.  We  now  put  our  specimen  on  a  thin  glass  plate 
(the  slide) ,  and  cover  it  with  a  still  thinner  small  glass  plate  (the  COVer- 
glass).  When  using  high-power  objectives,  the  latter  must  only  be  a  fraction 
of  a  millimetre  in  thickness,  on  account  of  the  small  object  distance. 

As  all  these  manipulations  require  time  and  sometimes  a  good  deal  of  time, 
our  specimen  will  in  the  meantime  suffer  changes.  In  all  animal  organism  great 
changes  take  place  soon  after  death,  which  alter  its  original  structure  to  a 
high  degree,  and  which  occur  all  the  sooner  if  the  organism  has  been  finely 
divided  and  separated  from  its  component  structures.  The  question  thus 
arises,  how  may  we  delay  these  changes  or,  if  possible,  prevent  them,  i.e.,  fix 
our  specimen.  But  supposing  that  we  have  made  fine  transparent  sections  of 
a  kidney  and  fixed  them  in  their  original  structure,  we  will  meet  with  still 
another  difficulty.  The  various  tissues  comprising  the  specimen,  e.g.,  epithe- 
lium, connective  tissue,  nerve  tissue,  etc.,  differ  so  little  in  their  optic  proper- 
ties, that  their  differentiation  will  be  extremely  difficult  to  the  untrained  eye, 
If  not  impossible.  The  examination  will  therefore  be  materially  facilitated  by 
subjecting  the  preparation  to  a  dyeing  process,  i.e.,  by  staining  them. 

We  may  thus  divide  microtechnical  methods  into  three  great  groups, 
namely : 

1.  The  Method  of  Preservation. 

2.  The  Method  of  Making  Sections. 

3.  The  Method  of  Staining. 

For  all  these  methods  scores  of  more  or  less  complicated  instruments  have 
been  devised  during  the  last  fifty  years,  and  a  host  of  chemicals,  reagents  and 

24 


25 

dves  have  been  recommended.     We  shall  now  acquaint  ourselves  with  these,  at 
least  as  far  as  their  practical  value  for  us  is  concerned. 


METHODS  OF  PRESERVATION 

Observation  of  the 
Living   Object. 

The  preparation  of  a  specimen  is  not  always  as  unfavorable  and  difficult 
as  in  the  case  of  the  human  kidney.  Going  down  the  scale  of  organisms,  we 
find  thousands  of  forms,  which  are  so  minute  and  transparent  that  we  can 
put  them  on  our  slide  without  further  preparation  and  keep  them  there  alive 
under  observation,  for  hours  and  even  days.  But  also  higher  organisms,  even 
human,  can  be  cut  and  their  sections  kept  alive  and  studied  for  some  time.  For 
instance,  it  is  not  very  difficult  to  observe  living  human  blood  or  spermatic 
fluid  under  the  microscope. 

Indifferent  Liquid  Media. 

The  great  importance  in  these  observations  is  to  examine  the  object  under 
conditions  as  nearly  normal  and  natural  as  is  available.  Just  as  we  would 
examine  infusoria,  obtained  from  a  sweet-water  aquarium,  in  this  same  me- 
dium, we  likewise  proceed  with  human  blood  corpuscles,  i.e.,  we  do  not  put  them 
in  water,  where  they  would  instantly  undergo  h;emolysis,  but  select  as  a  medium 
either  blood  serum  or  at  least  a  liquid  of  similar  properties,  i.e.,  an  isotonic 
fluid.  Of  course,  care  must  be  taken  that  this  fluid  does  not  evaporate  and 
thus  become  concentrated.  If,  on  the  other  hand,  a  hypototlic  fluid  should 
be  selected,  for  example,  distilled  water,  osmosis  would  take  place,  the  liquid 
entering  the  corpusles  and  distending  them.  If  a  hypertonic  solution  was 
used  the  result  would  be  a  loss  of  fluid  from  the  corpuscles  with  consequent 
shrinkage. 

The  most  satisfactory  results  for  mammalian  tissues  arc  obtained  with 
blood  serum,  which  may  be  secured  by  centrifuging  cooled  blood,  or  with 
amraotic  fluid,  which  may  easily  be  saved  from  the  gravid  uterus  of  the  cow 
or  pig.  The  aqueous  humor  from  the  anterior  chamber  of  the  eyes  of 
larger  animals  may  also  be  utilized  for  this  purpose,  and  can  be  procured  by 
puncture  with  a  Pravaz  hypodermic  needle. 

Of  the  artificial  indifferent  media  the  normal  or  physiologic  saline 
solution  is  the  best  known.  It  should  contain  0.9%  of  table  salt  for  exami- 
nation of  mammalian  or  human  tissues.  A  still  better  solution  is  Ringer's 
fluid,  containing  Nad,  8.0  gms.,  KC1,  0.2  gm.,  NaHCO.,,  0.2  gm.,  and 
CaCL,  0.2  gm.  in  1,000  cm3  of  distilled  water. 

Granted  that  the  study  of  living  organism  is  of  great  importance  in  the 
physiological  and  biological  research  into  the  animal  body,  the  fact  remains 
that  morphology  deals  to  a  greater  extent  with  specimens  which  have  pre- 
viously been  killed  and  preserved  in  the  proper  manner. 

Significance  of  Fixation. 

Naturally  the  first  prerequisite  is,  that  the  tissues  are  preserved  as  nearly 
as  possible  in  their  original  structure  and  relation.  The  object  is,  therefore. 


26 

to  fix  the  structure,  and  the  methods  and  reagents  employed  for  this  purpose 
have  been  termed  fixation,  and  fixing  solutions  or  fixatives.  If  the  ob- 
ject is  thus  killed  in  a  state  truly  resembling  that  of  life,  it  can  easily  be  pre- 
served for  an  unlimited  time. 

In  order  to  familiarize  ourselves  with  the  fixation  of  animal  tissue,  it  will 
be  a  good  plan  to  branch  off  for  a  short  excursion  into  physiologic  chemistry. 
The  cells  and  the  intercellular  substance  composing  the  body  tissue,  consist 
chiefly  of  water,  minerals,  carbohydrates,  fats  and  albumins.  The  latter  are 
for  us  the  most  important  of  the  constituents  of  tissues,  high-molecular,  very 
complicated  compounds,  which  are  partly  neutral,  partly  acid  and  partly  basic 
in  reaction,  being  soluble  in  water,  weak  alkalies  or  acids.  As  far  as  micro- 
technique is  concerned,  their  most  important  property  is  that  they  are  made 
insoluble  {coagulate),  when  treated  with  certain  physical  or  chemical  agents. 
One  of  the  best  known  and  most  common  physical  coagulating  agents  for 
albumin  is  heat.  If  an  albumin  solution  is  heated  to  nearly  100°,  all  the 
albumin  becomes  coagulated,  provided  the  solution  be  slightly  acidified.  The 
albumin  now  has  become  insoluble  in  its  former  solvent,  it  is  denatured.  If  a 
mineral  acid  or  certain  organic  acids  are  added  to  an  albumin  solution, 
coagulation  will  result  with  the  production  of  an  acid  albumin,  which,  how- 
ever, is  soluble  in  the  diluted  acid.  Another  important  group  of  substances 
coagulating  albumin  consists  of  the  salts  of  the  heavy  metals.  These 
change  the  albumin  into  metal  albuminates,  which  are  insoluble  in  water,  i.e., 
loose  compounds  of  metal  oxides  with  albumin.  Alcohol  also  deserves  men- 
tion as  a  coagulating  agent.  If  an  albuminous  solution  is  treated  with  alcohol, 
the  albumin  coagulates,  again  becoming  soluble  when  water  is  added.  In  this 
case,  therefore,  it  is  not  denatured.  If,  on  the  other  hand,  the  alcohol  is 
allowed  to  act  for  a  longer  time,  it  also  will  render  the  albumin  insoluble. 
Formalin,  a  solution  of  formaldehyde  in  water,  of  late  more  widely  known  in 
science  and  practice,  forms  methylcne  compounds  with  most  albumins,  which 
compounds  are  insoluble  in  water. 

This  property  of  albuminous  substances  is  of  great  interest  in  microscopy, 
enabling  us  to  fix  the  inconstant  tissue  picture  by  coagulating  the  otherwise 
soluble  albumins. 

Thus,  fixing  means  for  us  nothing  more  than  the  coagulation  of  albuminous 
bodies,  wherefore  every  good  fixing  agent  must  be  a  good  coagulant  of  albu- 
mins. JSut  again,  an  efficient  albumin  coagulator  need  not  be  a  good  fixing 
reagent ;  e.g.,  tannic  acid,  while  causing  albumin  to  coagulate  readily,  is  totally 
useless  as  a  fixative. 

Practical  experience  during  the  last  fifty  years  has  demonstrated  to  us 
many  methods  and  reagents,  which  might  be  used  for  fixation,  but  we  have  also 
learned  that  it  is  often  of  advantage  not  to  depend  upon  a  single  reagent  for 
fixation,  but  to  combine  the  good  properties  of  a  number  of  them ;  thus  alcohol 
or  metal  salt  solutions  are  used  in  conjunction  with  an  acid,  commonly  acetic 
acid,  each  of  which  would  yield  poor  results  if  used  alone. 

General  Rules  for  Fixation. 

Before  proceeding  to  the  special  discussion  of  the  various  fixatives,  it 
might  be  well  to  dwell  on  some  general  and  practically  important  points.  If  a 


27 

true  reproduction  of  our  preparation  is  desired,  it  follows  that  we  must  fix 
our  specimen  in  the  living  state,  which  proves  to  be  quite  a  difficult  task. 
When  dealing  with  animals,  the  living  object  is  more  often  available  and  the 
specimen  can  be  taken  directly  after  the  animal  has  been  killed  and  may  imme- 
diately be  put  in  the  fixing  solution.  With  human  materiaF  the  conditions 
are  rarely  so  favorable. 

Anesthetizing  and 

Killing   of  the  Animals. 

Most  commonly  chloroform  is  used  in  the  killing  of  animals.  Smaller 
animals,  up  to  the  size  of  a  small  dog,  should  best  be  put  under  an  adequately 
large  glass  globe  or  bell  glass.  A  tuft  of  cotton,  saturated  in  chloroform,  is 
introduced,  the  animal  carefully  watched  and  the  glass  held  fast,  since  many 
animals,  foremost  among  them  the  cat,  exhibit  a  state  of  tremendous  excite- 
ment. When  the  animal  has  become  motionless  and  reactions  have  ceased,  it 
is  attached  to  an  operating  board ;  the  thorax  is  quickly  opened  and  death  is 
brought  on  by  removing  or  opening  the  heart.  Larger  dogs  better  receive  an 
initial  dose  of  morphine  (6  cm3  of  a  1%  solution  of  morphine  muriate)  sub- 
cutaneously  and,  after  half  an  hour  has  elapsed,  will  easily  succumb  to  chloro- 
form administered  on  a  cloth.  A  very  convenient  and  humane  method  of 
killing  smaller  animals  is  the  administration  of  carbonic  add  by  means  of 
the  so-called  carbonic  acid  bomb,  whence  the  gas  is  led  through  a  tube  into  the 
bell  glass.  In  small  animals  like  frogs,  mice,  rats,  etc.,  death  is  generally 
induced  by  cutting  off  their  heads  with  strong  scissors.  When  using  a 
frog,  it  is  best  to  destroy  the  Spinal  cord  in  addition,  employing  a  medium 
sized  probe  for  this  purpose.  Another  very  useful  but  dangerous  agent  is  a 
2%  alcoholic  solution  of  hydrocyanic  acid,  which  is  kept  in  stock  in 
most  drug  stores.  A  few  drops,  injected  subcutaneously,  kill  even  larger  ani- 
mals almost  instantaneously. 

Specimens  from  the 
Human  Body. 

In  the  examination  of  human  material  much  time  is  often  lost  before  it 
reaches  us,  unless  we  are  dealing  with  an  operative  case  or,  perhaps,  an 
autopsy.  Material  taken  two  to  three  days  post  mortem  is  utterly  useless  for 
our  purpose.  The  maximum  time  which  may  elapse  between  death  and  the 
taking  of  a  section  is  twelve  hours.  A  very  efficient  and  easily  obtainable  mate- 
rial, which  serves  well  for  most  purposes,  is  the  body  of  a  child  during  the  first 
few  weeks  after  birth. 

Size  and  Treatment 
of  the  Specimen. 

Since  the  penetrating  power  of  most  of  our  fixing  agents  is  moderate,  it 
is  best  to  use  small  or  at  least  thin  pieces,  unless  other  reasons  make  this  im- 
practicable. A  section  of  1  cm  thickness  might  be  considered  as  very  thick. 

The  selected  section  should  not  be  handled  unnecessarily  with  instruments, 
and,  in  order  to  avoid  any  possible  crushing,  the  section  should  only  be  made 
with  sharp  instruments,  scissors  or  razor. 

The  amount  of  fixing  solution  should  be  plentiful,  the  volume  being  at 
least  twenty  times  that  of  the  object  to  be  fixed. 


28 


To  allow  free  access  of  the  fixative  from  all  sides,  the  specimen  is  best  sus- 
pended in  the  solution,  or  else  the  floor  of  the  jar  should  be  covered  with  glass 
wool,  cotton  or  several  layers  of  tissue-paper. 

Duration  of  Fixation. 

The  duration  of  the  fixing  process  naturally  depends  on  the  size  of  the 
object  and  the  nature  of  the  fixing  agent.  A  general  rule  may  be  adopted  that 
the  process  should  not  last  longer  than 'is  necessary  for  complete  saturation  of 
the  specimen.  The  speediest  result  is  obtained  from  acids ;  alcohol  and  formalin 
taking  slightly  longer,  while  the  metal  salts  work  slowest  of  all ;  e.g.,  after 
acting  four  hours,  a  5%  nitric  acid  solution1  will  have  penetrated  6  to  7  mm, 
10%  formalin,  5  to  6  mm;  96%  alcohol  3  to  4  mm,  0.3%  platinum  chloride 
only  0.5  to  1  mm.  Only  a  few  fixing  solutions  can  act  on  a  specimen  for  an 
indefinite  period  without  injuring  it,  i.e.,  can  also  serve  as  a  preservative.  Of 

these  we  may  mention  primarily  5  to  10%  forma- 
lin, in  which  a  specimen  can  remain  almost  un- 
changed for  an  indefinite  time.  To  a  certain  ex- 
tent this  can  be  said  of  alcohol,  which  should  be 
used  70  to  80%  strong.  All  other  agents  change 
the  specimens  under  prolonged  use ;  they  partly 
render  them  hard  and  brittle,  or  partly  deposit 
themselves  in  crystal  form,  thus  rendering  the 
staining  difficult,  if  not  impossible. 


PIG.  18. 

Jar  for  Washing  of  Fixed 
Specimens. 


After-Treatment. 

We  find,  therefore,  that  after  most  fixation 
methods  we  must  seek  to  extract  the  fixing  solu- 
tion after  it  has  done  its  work.  This  is  generally 

done  by  means  of  a  wash-glass  (Fig.  18),  which  is  suspended  on  the  knee- 
shaped  limb  in  a  water  reservoir,  fed  from  the  faucet.  Our  specimen  thus  lies  in 
a  continuous  current  and  within  a  few  to  twenty-four  hours  the  fixative  will  be 
entirely  washed  out.  Some  of  our  solutions  cannot  be  treated  that  way,  and 
foremost  among  them  are  the  acids.  Here  we  can  use  70%  alcohol,  which  must 
be  renewed  frequently,  or  5%  formalin. 

We  will  now  proceed  to  discuss  a  number  of  the  most  important  fixing 
solutions  which  are  of  interest  to  us. 

1.  Nitric  add,  HNO3,  when  pure  and  concentrated,  is  a  colorless  liquid 
of  1.54  specific  gravity;  when  exposed  to  air,  it  soon  assumes  a  yellow  color, 
due  to  the  formation  of  nitrous  acid.  The  commercial  concentrated  nitric  acid, 
which  is  generally  used,  contains  68%  HNO3  and  has  a  specific  gravity  of 
1.41.  The  C.  P.  nitric  acid,  obtained  at  druggists,  contains  only  25%  of 
HNO3,  having  a  specific  gravity  of  1.15.  When  nitric  acid  is  mixed  with 
alcohol,  the  main  compounds  formed  are:  Nitric  acid-ethyl  ether  (C2 
H5O.NO2),  nitrous  acid-ethyl  ether  (C,H,.ONO)  and  acctaldehyde 
(CH3.CHO).  It  must  be  borne  in  mind  that  the  ethers  of  nitric  acid  are 
quite  explosive  compounds  under  certain  conditions. 


1  When  speaking  of  any    per  cent,   solution,  we  naturally  always  mean  that  per 
cent,  solution  in  distilled  water. 


29 

Nitric  acid  itself,  or  its  compounds,  is  one  of  our  most  valuable  fixing 
agents,  and  especially  for  certain  organs,  e.g.,  the  retina,  it  cannot  be  excelled. 
It  is  mostly  used  in  a  7.5%  solution,  i.e.,  7.5  cm3  nitric  acid  of  1.41  specific 
gravity  and  92.5  cm3  of  water.1  This  agent  penetrates  the^  tissues  very  well, 
indeed  better  than  any  other  fixative.  Nitric  acid  must  not  be  allowed  to  act 
too  long  on  tissues,  as  it  will  otherwise  become  a  solvent  of  certain  elements 
of  the  cellular  tissue — five  to  six  hours  may  be  considered  the  maximum, 
although  ordinarily  three  to  four  hours  will  suffice.  If  the  object  should  be 
removed  from  the  acid  and  directly  placed  in  water,  a  swelling  of  the  connective 
tissue  will  take  place,  wherefore  the  acid  must  first  be  taken  out  with  other 
solvents,  e.g.,  70%  alcohol,  5%  solution  of  Glauber  salt,  or  5  to  10%  formalin. 

2.  Acetic   add,    CH3.COOH,    a    colorless    liquid    of    intensely    pungent 
odor.      Specific  gravity,  1.05.     It  crystallizes  at  about  70°   F.,  whence  it  is 
often  called  ice-vinegar.     The  boiling-point  is  approximately  298°,  and  the 
acid  burns  with  a  blue  flame.     It  must  be  kept  in  a  well-corked  bottle,  since  it 
is  extremely  hygroscopic  when  exposed  to  air. 

By  itself  this  acid  makes  a  poor  fixative,  but  as  an  ingredient  of  other 
reagents  it  plays  an  important  role  by  virtue  of  the  rapidity  with  which  it 
diffuses  through  the  tissues,  rendering  them  acid  and  thus  preparing  them  for 
the  action  of  the  actual  fixing  agent.  The  vapors  of  acetic  acid  are  also  used 
for  fixation  in  certain  instances. 

3.  Trichlor-acetic  add,  CC13 — COOH,   a   compound   in   crystal   form, 
dissolving  when  exposed  to  air  and  having  caustic  properties. 

A  5%  solution  is  quite  a  useful  fixative,  acting  much  slower  than  nitric  acid, 
hence  a  specimen  must  be  allowed  to  remain  up  to  ten  or  even  twelve  hours. 
The  acid  is  easily  washed  out  with  95%  alcohol. 

4.  Picric  acid,   C8H2(NO2)3.OH,    a  yellow  crystalline  powder,  slightly 
soluble   in  water   (1.1%),  more   readily  in   alcohol,   forming   a  bright  yellow 
solution. 

Picric  acid  solutions,  which  are  strongly  poisonous,  do  not  readily  pene- 
trate the  tissues  and  per  se  are  of  little  significance  for  our  purpose.  On  the 
other  hand,  picric  acid  becomes  a  potent  factor  when  forming  a  constituent  of 
a  solution,  and  is  especially  important  as  a  stain. 

5.  Chromic  acid,  Cr  O3,  a  reddish  brown  crystalline  compound,  soluble 
in  damp  air.      It   is   strongly  caustic   and  is   reduced  by   alcohol  into   brown 
chrome  hyperoxide,  respectively  green  chrome  oxide.     Chromic  acid  solutions 
should  not  be  filtered  through  paper. 

The  acid  is  used  in  a  0.25  to  1%  solution,  but  rarely.  Its  penetrating 
power  is  low,  although  greater  than  that  of  picric  acid.  The  object  is  usually 
left  in  the  solution  for  twenty-four  hours,  after  which  it  is  washed  in  running 
water  for  an  equal  length  of  time.  Any  precipitate  which  may  have  formed  in 
the  tissues  can  be  removed  by  treating  the  section  with  a  5%  solution  of 
potassium  cyanide.  An  objection  to  the  use  of  chromic  acid  lies  in  the  fact 
that  specimens  so  treated  become  brittle  and  are  stained  with  difficulty. 

6.  Osmium   tetroxid,   Os  O4 — incorrectly   named   osmic  acid,   is  manu- 

1  When  speaking  of  water  we  invariably  mean  distilled  water;  if  well  or  tap 
water  should  be  used,  it  will  be  specified  as  such. 


30 

factured  in  the  form  of  slightly  yellow  crystals,  which  arc  put  up  in  small 
glass  tubes  of  0.5  or  1.0  gr,  on  account  of  its  great  diffusibility.1  Its  vapors 
arc  very  irritating  to  the  mucous  membranes.  After  thoroughly  cleansing  the 
tubes,  they  are  filed  on  one  side  and  opened  by  touching  them  with  a  glass 
rod  heated  to  white  heat.  In  order  to  waste  nothing,  the  entire  tube  with 
contents  is  put  in  an  absolutely  clean  bottle  and  corked  with  a  tightly 
fitting  glass  stopper.  The  necessary  amount  of  water  can  now  be  added — 3 
to  4%  will  make  a  saturated  solution.  The  solution  must  be  protected  from 
dust. 

Osrnic  acid  has  attained  great  importance  in  microtechnique  owing  to  its 
properties  as  a  fixing  agent.  Although  only  moderately  able  to  coagulate 
albumin,  it  makes  an  excellent  fixative  for  the  cell,  particularly  the  cell  body. 
It  is  used  in  0.5  to  2%  solution,  or  in  the  form  of  vapor.  Thin  objects  are 
exposed  to  the  vapors  of  a  20%  solution  for  a  few  minutes.  Osmic  acid  solu- 
tions penetrate  the  tissues  only  slowly,  although  not  as  slowly  as  is  commonly 
stated.  Two  to  three  millimetres  are  saturated  in  a  few  hours.  The  staining 
of  osmic  acid  preparations  is  not  so  unsatisfactory,  as  is  often  supposed,  pro- 
vided that  the  acid  has  been  thoroughly  washed  out  in  water  for  twenty-four 
hours,  and  that  it  has  not  been  treated  with  alcohol  afterward.  To  preserve 
such  specimens  5%  formalin  is  to  be  recommended. 

Another  very  important  advantage  of  osmic  acid  lies  in  the  fact  that  it  is 
reduced  to  different  degrees  by  animal  tissues,  thereby  furnishing  a  finely 
shaded  picture.  It  is  most  strongly  reduced  by  fat  and  some  deutoplasmatic 
bodies,  which  in  consequence  appear  totally  black,  while  the  remainder  of  the 
picture  shows  gray  and  grayish  brown  hues.  A  secondary  staining  process 
can  therefore  often  be  dispensed  with. 

To  decolorize  osmic  acid  preparations,  the  sections  are  best  treated  with 
an  alcoholic  solution  of  hydrogen  peroxide  (one  part  of  the  commercial  per- 
oxide to  5 — 6  parts  of  70%  alcohol). 

7.  Chrome-Osmio- Acetic   Acid. — A  mixture   of   15   cm3    of   chromic 
acid  (1%),  4  cm3  of  osmic  acid  (2%),  and  1    cm'3    of   acetic    acid,   which  has 
received  the  name  of  Flemming's  solution,  has  a  wide  range  of  usefulness  in 
microtechnique,  and  constitutes  one  of  our  best  fixing  solutions.     In  rapidity  of 
penetration  it  excels  chromic  acid  as  well  as  osmic  acid.     The  staining  proper- 
ties of  specimens  so  treated  are  excellent,  inasmuch  as  no  alcohol  is  used  for 
after-treatment.     The  section  is  usually  fixed  for  twenty-four  hours,  but  might 
be  left  in  the  solution  for  several  days  without  any  harm  resulting  therefrom. 
Careful  washing  in  running  water  for  twenty-four  hours  after  completion  of 
fixation  is  indispensable. 

8.  Potassium  Bichromate,  K2  Cr2  O7. — Red  trigonal  crystals,   10% 
soluble  in  water,  giving  a  faintly  acid  reaction.     Per  se  potassium  bichromate 
is  an  inadequate  fixing  agent,  since  it  is  not  able  to  fix  those  albuminous  parts 
of  tissues,  which  are  most  valuable  to  us.     It  penetrates  slowly  but  steadily.     If 
one  desires  to  use  this  reagent,  it  must  be  acidified  first,  and  this  can  be  accom- 
plished with  acetic  acid.     To  a  3%  solution  add  5%  of  acetic  acid  and  fix  for 

1  Osmic  acid  is  at  the  present  time  a  very  expensive  reagent,  selling  at  about 
$2.00-$2.25  per  gram. 


31 

twenty-four  to  forty-eight  hours,  after  which  the  section  must  be  washed  in 
running  water  for  the  same  length  of  time. 

9.  Mueller's  fluid,  a  reagent  formerly  used  extensively,  is  to  this  date 
a  very  efficient  fixing  solution.     To  prepare  it,  2.5  gms  of  potassium  bichro- 
mate and  1  gm  of  sodium  sulphate  are  dissolved  in  100  cm:{  of  water.     It  is 
especially  adapted  for  the  preparation  of  the  central  nervous  system  for  sub- 
sequent staining  of  its  nerve  fibres.     But  even  as  a  fixing  agent  solely  Mueller's 
solution  gives  better  results  than  the  bichromate  solution  alone,  which  is  prob- 
ably due  to  a  liberation  of  small  amounts  of  chromic  acid  by  the  addition  of 
the  Glauber  salt.     The  solution  is  allowed  to  act  for  days,  weeks  and  even 
months,  but  it  must  be  changed  as  soon  as  it  becomes  turbid.     Entire  human 
brains  used  to  be  treated  in  this  fashion  for  months,  a  procedure  which  has  to- 
day been  abandoned  as  coarse  and  primitive.     After  fixation  the  running  water 
bath  is  essential.     Staining  properties  of  Mueller  preparations  are  good  for 
the  usual  carmine  and  hamnatoxylin  solutions.     In  some  cases  the  preparation 
with  Mueller's  fluid  is  even  a  prerequisite.     The  chrome  salt  present  acts  as  a 
corrosive. 

10.  Potassium-Bichromate-Osmic   Acid. — Of  the  other  potassium 
bichromate  mixtures  the  only  one  of  interest  to  us  is  the  potassium-bichromate- 
osmic  acid,  usually  designated   as  Altmann's  mixture.      It   consists   of  equal 
parts   of  2.5%   potassium  bichromate  solution  and  2%   osmic   acid   solution. 
Small  pieces  are  fixed  in  it  for  twenty-four  hours  and  are  washed  equally  long 
in  running  water. 

11.  Corrosive  Sublimate,  HgCl2,  occurs  in  white  crystals,  soluble  in 
water  to  the  extent  of  7%,  25%  in  alcohol,  giving  an  acid  reaction.     The  solu- 
tion is  not  stable  and  easily  breaks  up  on  exposure  to  light  with  the  production 
of  calomel.     More  stable  compounds  can  be  produced  by  using  normal  saline 
solution  instead  of  distilled  water,  the  former  giving  us  a  neutral  bichloride 
solution  by  the  formation  of  a  double  salt  compound,  chloride  of  mercury- 
chloride  of  sodium.     If  the  tincture  of  iodine  is  added  to  a  sublimate  solution, 
a  decolorization  of  the  latter  with  the  production  of  mercury  iodine  takes  place. 

Corrosive  sublimate  energetically  coagulates  albumin.  Its  aqueous  solu- 
tions, which  are  acid  in  reaction,  reduce  the  genuine  albumins  to  insoluble  albu- 
minates.  Albuminates  furnished  by  the  mercury  chloride-sodium  chloride  solu- 
tion become  insoluble  only  on  addition  of  an  acid.  The  penetrating  power  of 
corrosive  sublimate  is  medium,  pieces  of  5  mm  thickness  being  completely  fixed 
within  four  to  five  hours;  2.5  to  5%  watery  solutions  are  used,  and  it  is  to  be 
recommended  to  make  your  solution  fresh  just  shortly  before  using  it.  Such 
recent  solutions  give  decidedly  more  constant  results  as  do  older  table  salt- 
sublimate  solutions.  The  solution  can  be  used  with  or  without  additional 
acidification. 

12.  Corrosive  Sublimate  with  Acids. — For  acidifying,  various  acids 
can  be  utilized.     One  to  two  per  cent,  acetic,  7.5%  nitric  or  10%  trichloracetic 
acid  all  work  well.      Specimens   should  not   remain  longer  than  necessary  in 
neither   the   pure   sublimate   nor   the   acidified   solutions.      For   small   sections 
one  hour  should  suffice ;  larger  specimens  may  remain  six  to  eight  hours,  but 
twelve  hours  should  never  be  exceeded,  as  the  shrinking  caused  by  this  solu- 
tion increases  with  the  time  consumed  in  fixing.     Washing  of  the  section  in 


32 

running  water  for  twelve  to  twenty-four  hours  should  always  be  practised 
with  sublimate  as  well  as  sublimate-acetic  acid  solutions,  while  the  remaining 
acid  mixture  methods  are  best  followed  by  strong  alcohol,  to  avoid  a  swelling 
of  the  connective  tissues.  Material  for  frozen  sections  should  be  washed  and 
placed  in  5%  formalin.  Corrosive  sublimate  always  causes  coagula  in  the  con- 
nective tissues,  which  cannot  be  removed  even  by  the  most  scrupulous  washing. 
They  can  be  removed  either  in  the  section  or  in  the  bulk.  The  former  is  done 
in  frozen  sections,  where  the  coagula  are  dissolved  by  placing  the  section 
overnight  in  a  5%  solution  of  sodium  sulphate  and  afterward  washing  it,  chang- 
ing the  water  frequently.  When  dealing  with  specimens  which  are  to  be  em- 
bedded in  paraffin,  the  coagula  are  best  removed  by  the  use  of  70  or  80% 
alcohol,  to  which  has  been  added  enough  of  the  tincture  of  iodine  to  give  it  a 
good  brown  color.  The  iodide  of  mercury  is  thus  formed,  which  is  soluble  in 
alcohol,  giving  us  a  colorless  solution.  If,  therefore,  a  zone  of  colorless  alcohol 
is  found  around  the  specimen  on  the  following  morning,  the  process  must  be 
repeated,  until  no  more  decolorization  is  observed. 

Corrosive  sublimate,  despite  the  fact  that  of  late  it  has  lost  some  of  its 
reputation,  still  remains  one  of  our  very  best  fixing  agents;  objects  prepared 
by  its  use,  aside  from  excellent  preservation  of  structure,  show  great  staining 
properties. 

13.  Corrosive  Sublimate  -  Mueller's  Fluid  -  Acetic  Acid  Combi- 
nation,  known  as  Zenker's  fluid. — To  100  cm3  of  hot  Mueller's  fluid  5  gins  of 
bichloride  of  mercury    are  added,  and  just  before  use  this  solution  is  acidified 
with  3"  cm3  of  acetic  acid.     Duration  of  action  and  after-treatment  are  the 
same  as  with  corrosive  sublimate,  but  the  staining  properties  of  the  latter  are 
far  superior. 

14.  Corrosive  Sublimate -Chrome 'Osmium- Acetic  Acid. — To 
100  cm3  of  a  1%  bichloride  solution  we  add  10  cm?>    of    a    10%    chromic    acid 
solution,  5  cm3  of  a  2%  osmic  acid  solution  and  2  cm3  of  acetic  acid.     Duration 
of  fixation  is  twenty-four  hours,  to  be  followed  by  washing  in  running  water 
for  an  equal  length  of  time. 

15.  Platinum  Chloride. — The  product  sold  under  this  name  is  really 
platinum  chloride — hydrochloric  acid,  Pt  Cl4+2  H  Cl-)-6  H2  O;  it  forms  red- 
dish brown  crystalline  masses,  which  are  highly  soluble  in  water,  making  a 
strongly  acid  solution.     Owing  to  the  fact  that  this  compound  is  acted  on  by 
light,  it  is  best  to  keep  it  in  well-corked  brown  bottles. 

16.  Platinum  Chloride-Osmium-Acetic  Acid.  —  Platinum   chloride 
acts  well  on  albumin  and  therefore  is  an  excellent  fixing  agent ;  however,  it 
possesses  little  penetrating  power.     It  is  generally  made  use  of  in  the  form  of 
platinum  chloridc-osmium-acetic   acid  or  Hermann's  fluid,  which   consists   of 
15  cm3  of  1%  platinum  chloride,  4  cm3  of  2%  osmic  acid,  and  1  cm3  of  acetic 
acid.     In  its  action  it  -is  very  similar  to  Fleming's  solution.      Specimens  are 
fixed  from  twenty-four  hours  to  several  clays,  and  then  washed  for  twenty-four 
hours.     The  staining  properties  are  not  as  good  as  they  are  obtained  from 
Fleming's  solution. 

17.  Alcohol. — Ordinary  ethyl  alcohol,  C.,  H5  OH,  when  free  from  water, 
is  an  excellent  fixative  of  albumin  and,  if  allowed  to  act  long  enough,  furnishes 
durable  denaturation  products.     At  the  same  time  it  dehydrates  the  specimen, 


33 

so  that  the  section  may  be  rendered  free  from  water  by  the  repeated  use  of 
alcohol.  Alcohol,  when  judiciously  used,  is  a  very  good  and,  as  we  will  see 
later,  a  very  convenient  fixative.  As  regards  its  penetration  alcohol  takes  a 
medium  rank,  ranging  somewhere  after  potassium  dichromate  in  speed.  The 
staining  properties  are  good,  although  they  are  generally  overestimated.  A 
good  fixation  can  only  be  obtained  from  absolute  or  at  least  very  strong  alco- 
hol, and  here  again  we  find  a  disadvantage  in  that  the  water  is  extracted  too 
quickly  from  the  section,  so  that  delicate  moist  tissues  are  apt  to  shrink 
enormously. 

The  so-called  absolute  commercial  alcohol  contains  99.4 — 99.6%  of  pure 
alcohol,  which  is  absolutely  sufficient  for  our  purpose.  It  must  be  kept  in 
well-corked  bottles,  since  it  takes  up  water  assiduously  from  the  atmospheric 
moisture. 

Large  quantities  should  be  used  and  the  sections  suspended  freely ;  in  that 
fashion  it  will  penetrate  8 — 10  mm  within  twelve  hours.  If  a  specimen  is  left 
for  several  days,  it  becomes  very  hard;  it  is  well,  therefore,  to  transfer  it  to 
70 — 80%  alcohol  for  the  purpose  of  preserving  a  section.  After  a  time  the 
staining  property  of  such  preparations  is  materially  decreased. 

18.  Alcohol  Acetic  Acid. — Combinations  with  alcohol  are  innumerable, 
all  designed  for  counteracting  its  shrinking  property.     The  most  important 
adjuvant  is  acetic  acid.     One  part  of  the  latter  is  added  to  4  parts  of  alcohol 
(absolute),  the  mixture  is  allowed  to  act  for  twenty  minutes,  after  which  the 
section  is  transferred  to  a  mixture  of  1  part  of  the  acid  to  8  parts  of  alcohol, 
and  hence  to  pure  alcohol,  which  must  be  renewed  after  a  few  hours.     Of  the 
remaining  alcohol-acetic  acid  mixtures  Camay's  Is  the  best  known.     It  con- 
sists of  60  parts  of  absolute  alcohol,  30  parts  of  chloroform  and  10  parts  of 
acetic   acid.      It   penetrates   well   and   furnishes    a   good   nuclear   and   plasma 
fixation.     Fifteen  to  twenty  minutes  for  ordinary,  an  hour  for  larger  pieces, 
suffices  for  fixation,  after  which  the  object  is  transferred  to  absolute  alcohol, 
which  must  be  changed  several  times  within  the  next  few  hours. 

19.  Formalin.- — Formalin  is  a  40%   solution  of  formaldehyde,  H.COH, 
in  water;  it  is  a  colorless  fluid  with  a  pungent  odor,  not  only  irritating  mucous 
membranes,  but  even  producing  an  itching  erythema  on  susceptible  skin.     When 
fresh,  formalin  is  neutral  in  reaction,  but  it  gradually  becomes  acid,  due  to  the 
liberation  of  formic  acid. 

As  previously  mentioned,  formalin  can  coagulate  a  large  number  of  albu- 
minous bodies.  It  possesses  a  good  penetrating  power  and  in  no  way  diminishes 
the  staining  properties. 

Formalin  is  used  in  10%  solution,  and  specimens  can  be  kept  in  this  fluid 
for  an  indefinite  period,  so  that  this  solution  or,  perhaps  better,  a  5%  strength, 
makes  a  good  preserving  agent  as  well.  After  fixation  specimens  can  be 
directly  transferred  to  diluted  alcohol,  since  it  is  not  necessary  to  wash  out 
the  formalin. 

While  formalin  cannot  be  considered  among  the  very  best,  it  certainly  is  a 
good  fixing  agent,  and  for  certain  tissues,  e.g.,  muscle,  it  excels  all  others. 
When  properly  used  it  does  well  in  almost  all  instances. 

20.  Formalin-Mueller's   Fluid. — Of   the   various    compounds    of    for- 
malin Mueller's  fluid  with  formalin,  in  the  proportion  of  9 :  1,  is  the  best  known. 


34 

By  the  addition  of  formalin  the  fixing  property  of  Mueller's  fluid  is  materially 
increased.  When  the  mixture  becomes  milky  it  must  be  changed.  Specimens 
should  be  fixed  for  twenty-four  to  forty-eight  hours,  washed  in  water  and  pre- 
served in  5%  formalin. 

21.  Formalin- Alcohol. — Combinations  of  alcohol  with  formalin  are 
also  used  extensively.  Absolute  alcohol,  containing  10%  of  formalin,  will  fur- 
nish a  mixture  which  shrinks  the  tissues  less  than  does  alcohol  alone.  Twenty- 
four  hours  are  allowed  for  fixation,  and  according  to  what  after-treatment  is 
desirable,  the  specimen  is  transferred  to  absolute  or  diluted  (70%)  alcohol. 


PREPARATION   OF   SECTIONS 

The  methods  of  making  microscopic  sections,  which  we  will  now  deal  with, 
all  have  in  common  the  object  of  reducing  the  voluminous  organs  of  the  body 
into  small,  thin,  transparent  sections,  which  allow  the  rays  of  light  to  pass 
through  them,  thus  giving  us  a  specimen  which  is  adapted  to  microscopic  ex- 
amination. This  end  may  be  attained  in  many  different  ways.  Specimens  can 
be  teased,  crushed,  chopped,  shaken,  cut,  etc.  All  these  methods  have  two 
objects  in  view:  either  it  is  desired  to  preserve  the  relationship  of  constituent 
parts  of  a  tissue,  or  to  dissolve  such  relation  by  dissociating  or  isolating  the 
component  factors.  The  former  purpose  is  best  accomplished  by  the  cutting 
method,  which  enables  us  to  make  sections  of  any  desired  thickness,  either 
of  the  entire  organ  or  of  smaller  parts  of  same.  The  various  cutting  methods 
are  by  far  the  most  important  and  therefore  the  most  thoroughly  understood 
means  of  preparing  a  specimen.  Aside  from  these,  the  dissociation  methods 
enjoy  some  of  our  interest  by  making  it  possible  for  us  to  study  the  indi- 
vidual forms  of  the  tissue  elements.  The  chop-method  takes  a  position 
midway  between  cutting  and  dissociation  methods,  and  may  be  used  with  ad- 
vantage in  some  cases. 


THE    DISSOCIATION    METHODS 

Most  tissues  resist  the  mechanical  dissociation  of  their  component  parts; 
e.g.,  it  is  impossible  to  separate  a  muscle  fibre  from  the  tendon  fibre  to  which 
it  is  attached,  both  being  united  by  a  cement  substance.  The  same  is  true 
of  many  other  tissues,  so  that  our  first  aim  will  be  to  dissolve  this  cement 
chemically,  after  which  the  mechanical  isolation  can  take  place  without  diffi- 
culty. 

To  accomplish  the  former,  so-called  isolation-  or  maceration-agents  are 
used,  of  which  we  have  quite  a  large  selection.  We  will  only  mention  those 
which  are  most  important  for  our  purpose. 

Natural  (Spontaneous)  Maceration  After  Death. — After  tissue 
death  the  tissue  juices  furnish  a  medium,  which,  for  instance,  dissolves  the 
cement  substance,  gluing  together  the  epithelial  tissue.  For  this  reason  we 
find  that  in  objects  which  are  fixed  only  after  the  elapse  of  some  time  after 
death  the  epithelial  lining  has  been  stripped  from  the  underlying  tissue. 


35 

Alcohol. — A  fine  isolation  agent  for  epithelium  is  found  in  dilute  alcohol, 
1  part  of  90%  alcohol  to  2  parts  of  water.  Epithelial  tissues  may  easily  be 
macerated  after  twenty-four  hours. 

Acetic  Acid  Vapors. — Better  still  are  acetic  acid  vapors.  The  mucous 
membrane  in  question  is  spread  on  a  wax  or  glass  plate,  with  which  we  cover 
a  suitable  bowl  containing  a  small  amount  of  acetic  acid.  In  a  few  minutes  the 
epithelium  can  be  removed  in  large  pieces  from  the  basement  membrane. 

Potassium  Hydroxide  enjoys  quite  a  reputation,  and  is  prepared  by 
adding  to  32.5  gms  of  caustic  potash  67.5  cm3  of  water.  The  mixture  is 
agitated  well  in  a  beaker  until  the  potash  is  entirely  dissolved.  Since  much 
heat  is  generated  in  this  process  the  glass  beaker  must  be  kept  in  cold  water. 
This  strong  solution  will  isolate  non-striated  and  striated  muscle  fibres  in 
fifteen  to  thirty  minutes,  but  shrinks  such  tissues  considerably. 

Hydrochloric  and  Nitric  Acids. — For  the  isolation  of  the  tubules, 
ducts  and  alveoli  of  different  glands,  concentrated  mineral  acids  are  by  far  the 
best,  such  as  nitric  acid  and  hydrochloric  acid,  which  are  allowed  to  act  on 
thin  sections  for  from  twelve  to  twenty-four  hours. 

Mechanical  Maceration. — After  tissues  have  thus  been  prepared  by 
chemical  agents,  they  can  be  isolated  mechanically,  e.g.,  after  epithelia  have 
been  treated  by  the  above-mentioned  alcohol,  they  are  thoroughly  shaken  in 
their  container  and  the  coarse  pieces  of  tissue  are  removed.  After  a  few  hours 
the  cells  will  settle  on  the  bottom  of  the  glass  and  can  be  taken  out  with  the 
pipette.  Sedimentation,  however,  is  better  and  quicker  accomplished  by 
centrifuging. 

When  a  mucous  membrane  has  been  prepared  with  acetic  acid  vapors, 
small  pieces  of  the  epithelial  lining  are  picked  up  with  a  scalpel  or  spatula,  and 
the  cells  thereafter  isolated  on  the  slide  with  teasing  needles  or  by  stirring. 

Muscle  treated  with  potassium  hydroxide  solution  is  transferred  to  pure 
glycerine  and  tincture  of  iodine  is  added  until,  on  shaking,  the  fluid  ceases  to  be 
decolorized.  The  next  day  the  specimen  can  be  transferred  to  water  and  the 
fibres  separated  by  vigorous  shaking. 

When  dealing  with  glands  subjected  to  mineral  acids,  small  pieces  are  taken 
and  spread  carefully  on  the  slide  in  thin  glycerine  (1  part  of  glycerine  to  2 
parts  of  water). 

THE    CHOPPING    METHOD 

The  Chopping  Method. 

Although  somewhat  primitive,  this  method  yields  excellent  results  and  has 
the  advantage  of  being  less  destructive  to  the  tissues  than  other  methods.  It 
is  especially  indicated  in  the  examination  of  the  retina  and  striated  muscle,  no 
matter  whether  a  fresh  or  a  fixed  specimen  is  used.  It  can,  however,  be  em- 
ployed for  other  tissues  and  organs. 

A  small  piece  of  the  organ  is  placed  on  the  slide,  and  either  in  the  dry 
state  or  after  moistening  the  same  it  is  carefully  chopped  with  a  sharp  razor. 
Small  particles  will  be  found  to  adhere  to  the  knife,  and  must  be  removed  from 
time  to  time.  The  operator  must  take  care  not  to  spread  the  material  all  over 
the  slide,  but  aim  to  keep  it  as  closely  together  as  possible.  In  this  fashion 


36 

excellent  transverse  sections  of  the  retina  as  well  as  of  muscle  fibres  are  ob- 
tained with  the  additional  convenience  of  enabling  us  to  study  innumerable 
longitudinal  sections. 

THE    CUTTING    METHODS 

Cutting  of  the  Fresh  Specimen. 

As  observed  in  the  dissociation  method  of  fresh  specimens,  it  is  also  diffi- 
cult to  make  cut  sections,  unless  the  material  has  previously  been  subjected 
to  a  hardening  process.  An  object  must  possess  a  considerable  degree  of  hard- 
ness to  make  a  thin  section  possible,  and  in  the  natural  state  tissues  are  en- 
dowed with  very  little  stability,  excepting  very  few.  Either  they  are  much  too 
soft  and  do  not  furnish  enough  resistance  to  the  knife,  as  is  generally  the  case, 
or  the  deposits  of  lime  salts  make  them  so  hard  that  the  knife  is  unable  to 
penetrate  (e.g.,  bone,  tooth,  cartilage  of  older  individuals).  A  really  suitable 
consistency  in  man  and  the  mammalia  is  found  only  in  the  cartilage  of  the 
young. 

Cutting  of  the  Fixed 
and  Hardened  Specimen. 

When  dealing  with  a  fixed  specimen  the  prospects  are  much  more  favorable, 
most  fixing  agents  acting  to  a  certain  degree  in  a  hardening  capacity.  It  is 
for  that  reason,  that  fixation  and  hardening  are  often  used  promiscuously.  This 
is  not  entirely  justified,  since  we  have  fixing  solutions  which  not  only  fail  to 
harden  the  specimen,  but,  under  protracted  use,  even  decrease  their  consist- 
ency, at  the  same  time  fulfilling  all  their  requirements  as  fixatives.  The  best 
hardening  is  obtained  from  the  prolonged  use  of  alcohol,  which  is  for  this 
reason  often  continued  after  the  fixation  has  been  completed ;  e.g.,  alcohol 
formerly  was  extensively  and  to-day  is  to  some  extent  used  for  preparation  of 
the  central  nervous  system. 

Frozen  Sections. 

Cuts  obtained  in  the  said  fashion,  by  hardening  with  alcohol,  do  not,  how- 
ever, fulfil  all  the  requirements,  and  the  necessity  arises  to  find  other  more 
efficient  means  for  the  hardening  of  our  objects.  The  oldest  method,  known 
for  nearly  a  hundred  years,  is  freezing.  The  specimen  is  frozen  and  sections 
are  then  made.  This,  the  oldest  hardening  process,  is  used  to  this  day  with 
good  results. 

Embedding. 

In  this  method  our  specimen  is  saturated  with  a  substance,  originally  solid, 
which  has  been  liquefied  in  a  certain  manner  and  will  afterward  again  enter  into 
the  solid  state.  Of  these  substances  paraffin  and  celloidin  play  the  most  im- 
portant role  to-day.  To  embed  a  specimen  in  paraffin,  the  latter  must  be 
heated  to  above  its  melting-point.  The  specimen  immersed  in  this  molten 
paraffin  will  become  saturated  and,  after  cooling,  will  attain  a  consistency 
which  is  mainly  dependent  upon  the  melting-point  of  the  kind  of  paraffin  se- 
lected. Celloidin,  on  the  other  hand,  is  liquefied  by  dissolving  it  in  a  mixture  of 
alcohol  and  ether.  Here,  too,  the  specimen  is  immersed  in  the  solution,  and 


37 

hardening  is  secured  by  the  evaporation  of  the  solvent  and  suitable  after-treat- 
ment. 

Cutting  Instruments. 

Formerly  the  razor  was  used  exclusively;  it  requires,  however,  great  skill 
and  an  excellent  knife  to  follow  this  practice.  Hence  the  razor  is  not  to  be 
recommended  to  the  beginner.  Modern  times  have  evolved  instruments  which 
make  it  possible  for  him  to  make  useful  sections  without  special  skill.  Such 
instruments  are  designated  as  microtomes. 

Following  we  will  discuss  in  detail  the  three  most  important  cutting 
methods. 

Frozen  Section  Method 

It  is  a  well-known  fact  that  freezing  does  not  in  any  way  injure  the  greater 
number  of  tissues,  not  even  the  highly  developed  organisms.  We  need  only 
call  attention  to  the  experiments  of  Raoul  Pictet,  who  froze  frogs  to  a  tem- 
perature of  28°  C.  below  the  freezing-point,  so  that  they  were  as  hard  as 
glass,  and  yet  after  thawing  them  up,  the  animals  recuperated  and  lived. 
Muscle  of  mammalia  can  be  exposed  to  a  temperature  of  — 50°  C.  for  a  certain 
length  of  time ;  it  will  contract  vigorously  after  the  temperature  has  been 
brought  up  to  normal  again.  Freezing  fixed  specimens  does  not  deteriorate 
the  latter  in  the  least.  It  is  evident  therefore  that  the  freezing  process  fur- 
nishes an  excellent  and  unharmful  method  of  hardening. 

In  former  years  it  was  a  difficult  or  expensive  task  to  secure  the  low  tem- 
peratures necessary  for  the  freezing,  but  all  obstacles  have  been  surmounted 
by  the  simple  and  inexpensive  method  found  in  the  use  of  solid  carbonic 
acid. 

Preparation  of 
Solid  Carbonic  Acid. 

The  solid  acid  may  be  prepared  from  the  commercial  liquid  carbonic  acid 
in  the  following  manner :  Use  a  bag  made  of  strong  silk  velvet  or  chamois, 
which  is  open  on  one  side,  where  it  can  be  drawn  together  after  the  fashion  of 
a  tobacco-pouch.  After  taking  off  the  cap,  which  closes  the  carbonic  acid 
drum,  this  pouch  is  drawn  over  the  mouth  of  the  drum  and  secured  tightly. 
The  tank  is  placed  on  a  chair,  the  head  being  slightly  lowered,  and  the  valve  is 
opened  for  about  ten  to  fifteen  seconds.  After  removing  the  bag  we  will  find 
it  filled  with  a  light  white  snow,  the  solid  carbonic  acid.  The  snow  is  col- 
lected on  a  cloth  and  thence  transferred  into  a  wooden  container,  provided 
with  a  funnel,  and  here  it  is  ground  together  and  compressed  with  hammer  and 
rod  (Fig.  19  a).  After  a  few  seconds  a  cartridge  of  carbonic  acid  can  be 
pressed  from  the  wooden  container,  which  can  be  kept  for  hours  in  an  isolated 
glass  cylinder,  the  so-called  Dewar — or  thermos  bottle  (Fig.  19  fr). 

The  Freezing  Cylinder. 

To  freeze  a  specimen,  the  aim  must  be  to  guide  the  cold  furnished  by  the 
cartridge  exclusively  t'o  and  into  the  specimen.  To  accomplish  this  purpose  a 
freezing  cylinder  (Fig-  20)  is  used.  It  consists  of  a  metal  cylinder  (a), 
which  contains  a  Dewar  bottle  (c),  the  latter  being  protected  by  a  felt 


38 


coat  (d).  The  Dewar  bottle  is  a  double-walled  glass  cylinder,  the  walls  of 
which  are  silvered,  the  space  between  the  walls  having  been  reduced  to  a 
vacuum.  This  serves  to  reduce  the  loss  of  heat  by  conduction  and  radiation  to 
a  minimum.  The  metal  cylinder  is  closed  on  top  by  a  threaded  cover,  on  the 
under  surface  of  which  we  find  attached  a  second  metal  cylinder  (A1),  which 
projects  into  the  cavity  of  the  Dewar  bottle.  In  this  smaller  cylinder  the  car- 
bonic acid  cartridge  is  placed  from  below  and  the  cover  I  is  tightly  adjusted. 
To  the  inner  side  of  this  cover 
a  spiral  spring  is  attached  (TW) 
which  serves  to  press  the  car- 
bonic acid  tightly  against  the  HSUl—  /* 
undersurface  of  the  upper  large 
cover.  Into  the  latter  the  freez- 


FIG.  19. 

Press,  a,  and  Eetainer,  b,  for  Solid 
Carbonic  Acid 


Construction  of  Freezing  Cylinder. 
Explanation  in  text. 


ing-table  proper  is  screwed  (o),  being  separated  from  the  outer  metal  cylinder 
by  a  hard  rubber  ring. 

The -Freezing  Microtome. 

The  described  apparatus  can  be  adjusted  to  most  microtomes.  We  pre- 
fer an  instrument  furnished  by  M.  Schanze  (Leipzig),  which  combines  stability 
with  greatest  precision  and  is  not  very  expensive.  In  Fig.  21  we  see  the  in- 
strument with  the  freezer  attached.  It  rests  on  a  heavy  cast-iron  plate  {gpl}-, 
from  which  arises  a  vertical  plate  (vpl),  which  in  turn  gives  off  a  third  plate 
at  an  angle  of  45°.  This  latter,  together  with  the  upper  part  of  the  vertical 


39 

plate,  thus  forms  an  horizontal  track  in  which  glides  the  knife-carriage,  a 
heavy  triangular  metal  block  (mschl).  Its  upper  surface  serves  for  the  attach- 
ment with  bolt  and  nut  of  the  knife  (ra),  which  is  provided  with  a  forklike 
handle.  On  the  other  side  of  the  vertical  plate  (the  one  turned  toward  us) 
we  find  the  specimen-carrier.  It  glides  on  two  vertical  rails,  the  left  only 
being  visible  in  our  picture  (opl).  A  lever  (h)  serves  to  move  the  specimen- 
carriage  (oschl)  up  and  down.  In  close  approximation  with  the  specimen- 
carriage  is  the  specimen-holder.  It  consists  of  two  shafts,  placed  at  right 
angles  to  each  other,  which  can  both  be  secured  by  means  of  a  screw  (fschr). 
The  specimen-holder  proper  (oh)  is  connected  with  the  upper  shaft;  it  mainly 
consists  of  a  ring,  which  receives  the  freezing  apparatus  (gfr).  The  latter 
can  be  raised  or  lowered  in  the  ring  by  means  of  a  screw.  The  finer  adjust- 
ment of  the  specimen-carriage  can  be  procured  by  the  use  of  the  micrometer- 
screw.  It  is  only  partly  visible  in  our  picture,  the  lower  end  being  hidden  in 


mschr 


FIG.  21. 
The  Freezing  Microtome.    Paraffin  Clamp  at  the  Right  of  the  Microtome. 

the  clamp  (zw),  while  the  specimen-carriage  rests  on  its  upper  extremity.  The 
section  scale  (tsch)  serves  for  the  adjustment  of  the  thickness  desired.  It  is 
divided  into  50  whole  or  100  half  degrees,  respectively.  If  the  disc  is  turned 
one  whole  degree  toward  the  left,  the  specimen  will  be  raised  10  m  so  that  thick- 
nesses of  5  \L  and  its  multiples  can  easily  be  obtained. 

The  Practical  Use  of 
the  Freezing  Microtome. 

Unscrew  the  cover  of  the  freezing  apparatus,  which,  being  intimately  con- 
nected with  the  freezing  cylinder,  will  lift  the  latter  out,  then  open  the  lock- 
cover  on  the  lower  end  of  this  cylinder,  insert  a  cartridge  and  close  it  again. 
The  cover  is  now  screwed  on  again  and  the  specimen,  moistened  with  a  few 
drops  of  water  or  normal  saline,  is  placed  on  the  freezing-table.  After  five  to 
ten  minutes  it  will  be  entirely  frozen.  The  knife  should  be  attached  to  the 
knife-carriage  so  that  it  stands  vertical  to  the  long  axis  of  the  microtome.  The 
screw  designed  to  hold  the  knife  (mschr)  must  necessarily  be  drawn  tight.  By 
rotating  the  freezing  cylinder  up  and  down,  the  upper  surface  of  the  specimen 
is  closely  approximated  to  the  edge  of  the  knife,  the  apparatus  fixed  in  this 


40 

position,  and  we  can  now  proceed  to  cut.  The  knife-carriage  is  moved  for- 
ward with  the  right  hand,  evenly  and  without  exerting  any  pressure.  Preced- 
ing each  cut  the  left  hand  turns  the  section  scale  a  corresponding  number  of 
degrees  to  the  left,  according  to  what  thickness  may  be  required.  The  sections 
obtained  either  cling  to  the  knife,  becoming  soft,  or  they  drop  to  the  freezing- 
table,  where  they  will  lie  in  the  frozen  state.  All  these  manipulations  are  much 
easier  executed  than  they  are  described. 

How  to  Obtain  a 
Favorable  Consistency. 

The  consistency  of  our  specimen  is  naturally  of  prime  importance  for  the 
securing  of  useful  sections,  and  the  former  is  mainly  dependent  upon  tempera- 
ture, though  also  influenced  by  the  structure  of  the  specimen.  Our  apparatus 
produces  a  temperature  of  — 12  to  — 15°  on  the  freezing  plate.  It  is  generally 
used  for  fresh  specimens,  those  retaining  their  own  natural  tissue  juices;  for 
objects  that  have  been  taken  out  of  water  and  are  therefore  also  saturated  with 
it,  the  temperature  is  too  low,  since  such  objects  would  split  on  cutting.  This 
objection  can  be  obviated  by  placing  the  specimen  in  a  fluid  having  a  lower 
freezing-point  than  water,  e.g.,  very  dilute  alcohol  or,  better  yet,  dilute  for- 
malin, where  it  should  be  kept  for  several  hours.  The  author  recommends  5% 
formalin,  which  should  not  be  removed  before  placing  the  object  on  the  freez- 
ing-table. This  procedure  will  almost  invariably  give  good  results,  and  only 
very  seldom  will  one  have  to  resort  to  10%  formalin. 

Time  and  care  should  be  taken  in  cutting,  as  our  apparatus  will  keep  the 
specimen  frozen  for  at  least  one-half  hour,  and  if  signs  of  thawing  should  be 
noticed,  it  will  only  be  necessary  to  insert  a  fresh  carbonic  acid  cartridge,  and 
the  work  can  be  continued  in  leisure. 

Thickness  of  Sections. 

As  regards  the  thickness  of  sections,  we  can  easily  obtain  cuts  of  5 — 10  ;*, 
even  with  a  large  cutting  surface,  by  the  use  of  this  freezing  microtome ;  but 
we  do  not  recommend  such  thin  sections,  unless  especially  required,  since  the 
further  treatment  of  sections  becomes  more  difficult  the  thinner  the  cut. 
Sections  of  15 — 25  ^  thickness  should  answer  our  purpose  and  will  always  show 
even  the  finest  structural  details,  if  they  are  stained  in  the  proper  manner. 

Treatment  of  the   Sections. 

If  the  section  curls  up  and  drops  to  the  freezing-table,  the  transport  is 
very  simple ;  it  is  grasped  with  a  pair  of  fine  tissue  forceps  and  thrown  into  a 
bowl  of  water,  where  it  will  generally  unfold  itself  without  any  aid.  Should  the 
section  adhere  to  the  knife  and  thaw,  it  will  have  to  be  brushed  off  with  the 
tip  of  the  finger,  which  is  then  dipped  into  the  water.  Each  section  may  be 
taken  off  separately,  or  the  operator  may  wait  until  a  number  have  collected 
and  then  take  them  off  en  masse.  The  finger  should  be  thoroughly  dried  each 
time  to  avoid  bringing  water  to  the  cutting  surface. 

When  handling  a  very  small  and  thin  specimen  it  is  best  to  first  freeze 
enough  water  to  make  a  layer  of  ice  2 — 3  mm  in  thickness,  on  top  of  which  the 
object  is  placed.  If  we  want  to  remove  the  latter  again,  the  freezing-table  must 
be  unscrewed  from  the  cover  and  water  poured  on  its  reverse  surface  for  a  few 


41 

seconds,  or  the  whole  of  it  dipped  in  water,  after  which  the  specimen,  still 
frozen,  can  be  detached. 

The  Paraffin-Section  Method 

Much  more  complicated  than  the  freezing  is  the  paraffin-section 
method.  The  first  consideration  will  be  to  embed  the  specimen  in  paraffin, 
to  wit,  not  only  to  give  the  object  a  coating  of  paraffin,  but  to  saturate  every 
part  of  the  specimen.  This  is  practicable  only  if  the  specimen  is  first  thor- 
oughly penetrated  by  a  liquid,  which  is  a  good  solvent  of  paraffin,  and  for  this, 
reason  quite  a  complicated  preparatory  treatment  is  necessitated. 

Paraffin. 

As  paraffin  are  classed  mixtures  of  hydrocarbons  of  the  general  formula 
of  Cn  H2n  ^  2  in  which  the  value  of  n  fluctuates  between  20  and  27.  It  is  a 
coal-tar  product,  but  has  also  other  sources,  e.g.,  the  hardest  variety  has  its 
origin  in  ozocerite.1  It  is  a  white,  fatty  substance  of  a  specific  gravity  of  0.9. 
The  melting-point  may  be  anywhere  between  40°  and  85°  C.,  according  to  the 
origin  of  the  paraffin.  Paraffin  is  absolutely  insoluble  in  water  and  almost 
insoluble  in  cold  alcohol.  It  is  soluble  in  carbon  disulphide,  chloroform,  ben- 
zine, benzol,  xylol,  toluol,  petroleum  ether,  etc.  The  boiling-point  is  300°. 

Preparatory  Media. 

The  reader  will  notice  that  the  above-mentioned  solvents  of  paraffin  do  not 
mix  with  water  in  any  proportion,  but  can  easily  enter  into  a  mixture  with 
alcohol  in  any  proportion ;  if,  therefore,  we  want  to  saturate  our  watery  speci- 
men with  such  a  solvent  of  paraffin,  which  we  will  call  a  preparatory  me- 
dium, it  will  first  be  necessary  to  dehydrate  the  tissues  by  the  use  of  alcohol. 
Let  us  select  chloroform  as  a  standard  preparatory  medium,  since  it  pos- 
sesses many  advantages  over  the  others.  The  preparatory  treatment  will  be 
as  follows : 

Dehydration. 

The  fixed  and  washed  specimen,  which  we  suppose  to  be  in  water,  is 
gradually  transferred  to  alcohol  of  increasing  strength.  Starting  with  10% 
alcohol  we  increase  at  the  rate  of  10%.  We  make  the  desired  strength  by 
diluting  absolute  alcohol  with  the  corresponding  amount  of  water.  The  length 
of  time  required  for  each  grade  of  alcohol  depends  wholly  on  the  size  of  our 
specimen.  Pieces  of  1  cm3  will  require  approximately  twelve  hours,  while  two 
to  three  hours  suffice  for  smaller  pieces.  The  specimen  should  always  stay  in 
its  container,  for  which  a  wide-mouthed  bottle  will  serve  best ;  the  latter  should 
be  corked  and  labelled  as  to  origin  of  specimen,  manner  of  fixation,  and  the 
respective  alcohol  in  use.  In  this  fashion  specimens,  lest  they  be  very  large, 
can  be  dehydrated  in  five  days,  if  at  the  end  we  rechange  the  absolute  alcohol 
once. 

The  introduction  of  the  specimen  into  the  preparatory  medium  should  also 
take  place  gradually.  As  a  rule,  three  steps  will  suffice.  First,  3  parts  of 

1  £<>—  smell, —  KTip6s=  wax;  a  natural  paraffin,  found  in  Galizia,  having  a  green, 
brown  or  red  color  and  an  odor  of  petroleum. — (The  Translator.) 


42 


alcohol  are  mixed  with  1  part  of  chloroform,  then  equal  parts  are  used  and, 
finally,  3  parts  of  chloroform  to  1  part  of  alcohol.  In  each  of  these  mixtures 
the  specimen  stays  from  six  to  twelve  hours,  although  a  longer  period  will  do 
no  harm,  after  which  it  is  transferred  to  pure  chloroform  overnight. 

The  saturation  with  paraffin  can  only  be  effected  by  placing  the  specimen 
in  paraffin,  previously  heated  until  liquid,  and  by  keeping  the  same  liquid 
during  the  entire  process.  To  do  this  we  must  use  a  thermostatt  similar  in 
construction  to  those  used  for  culturing  bacteria  and  for  the  artificial  breeding 
of  chicken  eggs.  Fig.  22  represents  such  an  apparatus,  which  is  suitable 
for  our  purpose.  It  is  made  of  strong  copperplate,  has  double  walls  and  a 

door,  the  space  between  the  walls  being 
filled  with  water,  or,  better  still,  with 
acid-free  glycerine.  A  water-gauge  will 
indicate  the  depth  of  the  water.  A 
second  box  surmounts  the  former,  also 
equipped  with  a  door,  but  having  only 
simple  metal  walls.  The  temperature 
obtained  in  either  compartment  is  indi- 
cated by  separate  thermometers.  The 
heating  is  done  by  a  burner  placed  be- 
neath the  lower  box;  this  burner  has  a 
safety  device  which  shuts  off  the  gas 
supply  in  case  the  flame  should  suddenly 
be  extinguished.  Before  entering  the 
burner  the  gas  passes  through  a  regu- 
lator situated  within  the  double  wall. 
The  temperature  can  thus  be  regulated 
at  will,  so  that  only  so  much  gas  will 
be  admitted  to  the  burner  as  is  neces- 
sary to  obtain  the  desired  temperature. 
There  are  also  similar  thermostats  on 
the  market  which  can  be  heated  with 
petroleum  or  by  means  of  electricity. 
The  temperature  of  the  thermostat 

should  exceed  the  melting-point  of  the  paraffin  in  use  by  about  1 — 2°.  As  will 
be  shown  later,  we  select,  as  a  rule,  a  paraffin  having  a  melting-point  of  54 — 
56°,  and  should  therefore  regulate  the  temperature  of  our  thermostat  to 
56 — 58°.  This  temperature  will  be  maintained  in  the  lower  compartment, 
while  in  the  upper,  which  is  indirectly  heated  from  the  lower,  the  temperature 
obtained  will  range  between  40°  and  45°. 

The  Melting-Point 
of  Paraffin. 

An  important  factor  in  the  paraffin  method  is  the  correct  selection  of  the 
paraffin  as  regards  its  melting-point.  The  following  considerations  should  be 
borne  in  mind :  The  thinner  we  desire  the  sections  to  be  made,  the  harder  the 
paraffin  must  be,  or,  in  other  words,  the  greater  must  be  the  difference  between 
the  room  temperature  and  the  melting-point  of  the  paraffin ;  e.g.,  witli  a  paraffin 


FIG.  22. 

Thermostat  for  Embedding  in 

Paraffin. 


43 

of  52°  melting-point  sections  of  5  jx  or  even  thinner  can  be  made  in  a  cool 
room,  which  would  be  impossible  in  a  warm  room  or  on  a  hot  summer  day. 
Again,  in  the  former  case  it  would  be  hard  to  make  sections  as  thick  as 
25 — 50  [A,  which  would  be  an  easy  task  in  the  latter  instance.  We  might  say 
that  the  preparation  of  thin  sections  requires  a  differencc_between  room  tem- 
perature and  paraffin  melting-point  of  approximately  40°.  If  this  difference 
decreases,  thin  sections  will  be  compressed  during  the  cutting  process,  and  will 
consequently  be  deformed.  For  thick  cuts  the  difference  must  not  exceed 
30 — 35°.  If  it  is  raised  in  this  instance  the  section  will  break. 

The  best  plan  is  to  select  two  sorts  of  paraffin,  one,  the  soft  paraffin,  with 
a  melting-point  of  42°,  and  the  other,  the  hard  paraffin,  with  a  melting-point 
of  56°.  The  latter  is  used  for  thin  sections,  while  in  the  preparation  of  thicker 
sections  we  will  have  to  add  more  or  less  of  the  former  quality  to  the  latter. 
As  a  receptacle  for  the  paraffin,  round  dishes  with  a  flat  bottom,  lined  with 
porcelain  or  enamelled  tin,  will  serve  best. 

Saturating  the 
Specimen  with  Paraffin. 

Great  care  must  be  used  and  the  temperature  changes  must  be  gradual 
when  the  specimen  is  to  be  transferred  from  the  preparatory  medium  into  the 
liquid  paraffin.  After  the  specimen  has  been  saturated  with  chloroform,  the 
excess  of  the  latter  is  decanted  until  only  sufficient  is  left  to  cover  the  object; 
then  a  few  pieces  of  soft  paraffin  are  added  and  the  uncovered  glass  is  placed 
in  the  upper  compartment  of  the  thermostat.  After  a  few  minutes  (15  to  30) 
the  paraffin  will  dissolve  and  a  few  more  pieces  can  be  added,  and  so  on  until  a 
sufficient  quantity  of  paraffin  is  in  the  glass  to  cover  the  specimen,  while  in  the 
meantime  the  chloroform  is  slowly  evaporating.  The  latter  process  will  be 
complete  the  next  morning,  and,  instead  of  chloroform,  our  specimen  will  now 
be  saturated  with  the  liquid  soft  paraffin. 

During  this  time  we  can  prepare  our  liquid  hard  paraffin  in  the  following 
manner :  A  suitable  basin  is  filled  to  the  brim  with  small  pieces  of  hard  paraffin, 
and  placed  in  a  lower  compartment  of  the  thermostat.  During  the  night  the 
paraffin  will  be  liquefied.  The  specimen  is  now  brought  from  the  upper  division 
into  the  lower  and  after  reaching  the  temperature  of  58°  in  about  half  an 
hour,  it  is  transferred  to  the  hard  paraffin  with  a  heated  spatula.  It  should 
not  be  kept  there  unnecessarily  long,  but  not  less  than  two  hours,  when  reason- 
ably large.  Sudden  temperature  changes  are  deleterious,  more  so  than  too  pro- 
longed, but  gradually  completed  exposure  to  hard  paraffin. 

Paraffin  Embedding. 

This  brings  us  finally  to  the  embedding  pro- 
cess proper.  This  can  be  accomplished  in  little 
paper  boxes,  watch  glasses,  or,  better  than  either, 
in  small  metal  boxes.  They  consist  of  two  brass  j>.  23 

strips  bent  at  right  angles,  one  limb  being  shorter       Frame  for  Embedding  in 
than  the  other  (Fig.  23).     When  the  two  strips  are  Paraffin, 

brought   together  they   form,  with  the  underlying 

glass  plate,  a  little  box,  which  is  open  on  top  and  the  length  of  which  can  be 
adjusted  as  desired.   The  glass  plate  and  inner  surfaces  of  the  brass  are  thinly 


44 

coated  with  vaseline.  The  box  is  now  filled  with  paraffin  and  the  specimen  im- 
mersed in  the  same,  and  then  deposited  in  a  position  most  suitable  for  cutting. 
Several  specimens  can  be  embedded  in  this  manner  in  one  box  by  placing  them 
at  a  proper  distance. 

Cooling  of  the  Block. 

The  paraffin  must  needs  be  cooled  now,  and  this  can  easily  be  accomplished 
by  placing  the  box  including  the  glass  plate  in  a  large  basin  containing  cold 
water.  Care  must  be  taken  here,  since  the  lighter  paraffin  tends  to  rise  to  the 
water-surface.  This  can  be  overcome  by  carefully  inclining  the  bed,  and  let- 


FIG.  24. 

Position  of  Knife  in  the  Cutting  of  Paraffin.  Sections. 
I,  correct;  II,  too  acute;  III,  too  obtuse. 

ting  the  water  flow  over  it  gradually.    After  a  few  hours  our  block  is  completely 
solidified  and  can  easily  be  removed  from  the  bed. 

The  Microtome  for 
Paraffin  Sections. 

The  microtome,  mentioned  previously,  can  also  be  used  here;  we  simply 
remove  the  freezing-table  and  substitute  the  paraffin  clamp,  which  is  depicted 
in  Fig  21,  lying  next  to  the  microtome.  The  paraffin  block  itself  may  be 
properly  shaped  and  adjusted  in  the  clamp,  or  it  can  preferably  be  mounted 
first  on  a  small  wooden  block  and  the  latter  fastened  in  the  clamp.  Of  course 
the  clamp  can  be  left  out  all  together,  and  the  paraffin  block  molten  directly 
to  the  freezing-table. 

The  Cutting  of 
Paraffin  Sections. 

This  proves  more  difficult  to  the  beginner  than  the  preparation  of  frozen 
sections,  for  the  reason  that  in  this  method  the  position  of  the  knife  is  of 


45 

great  importance.  While  it  does  not  matter  whether  our  knife  is  in  an  oblique 
or  sloping  position,  the  angle  formed  by  the  cutting  edge  and  the  horizontal 
plane  is  the  vital  issue.  The  microtome  knife  is  wedge-shaped  and  acts  like 
a  wedge,  the  surfaces  becoming  abruptly  converging  near  the  cutting  edge,  thus 
practically  forming  a  composition  of  two  wedges,  the  two  converging  surfaces 
near  the  edge  receiving  the  name  of  cutting  facettes.  If  w-e  picture  an  object 
being  placed  before  this  double  wedge  (Fig.  24),  it  will  be  possible  to  make 
a  section  only  if  the  lower  facette  c  d  either  coincides  with  the  cutting  surface 
a  b  (I)  or  forms  with  it  an  angle  above  180°  (II).  If  the  angle  should  be  less 
than  180°  (III)  no  cut  will  result,  since  the  facette  will  simply  glide  over  the 
surface  of  the  object,  polishing  it  smooth.  Only,  if  the  specimen  is  elevated 
enough  to  let  its  surface  be  on  a  higher  level  than  the  knife-edge,  an  irregular 
section  may  be  accomplished.  To  make  a  correct  cut,  the  knife  must  be  rotated 
upon  its  long  axis  and  adjusted  more  steeply.  Larger  instruments  possess  for 
this  purpose  a  separate  knife-holder,  but  we  can  accomplish  the  same  purpose 
by  using  two  small  wooden  plates,  placing  one  under  the  anterior,  the  other 
over  the  posterior  handle  of  the  knife.  A  metal  ring  is  placed  on  top  and  the 
whole  fastened  tightly  in  the  clamp.  Several  sizes  of  wooden  plates  should  be 
kept  on  hand,  so  that  an  appropriate  thickness  may  be  selected,  since  the  angle 
of  the  cutting  facettes  changes  with  almost  every  honing  of  the  knife.  Again, 
the  position  may  be  too  steep ;  this  will  be  indicated  by  the  presence  of  numer- 
ous breaks  in  the  section,  running  parallel  with  the  edge  of  the  knife. 

The  Curling  of  Sections. 

All  paraffin  sections  have  more  or  less  tendency  to  curl  upon  themselves. 
This  must  be  obviated  by  the  use  of  a  camel's-hair  brush.  While  the  right 
hand  moves  the  knife-carriage,  the  left  holds  the  brush,  and  as  soon  as  the  knife 
enters  the  substance  of  the  block,  but  not  before,  the  section  is  gently  pressed 
against  the  knife. 

Mounting  of  Sections. 

The  next  step  is  the  mounting  of  the  section  on  the  slide,  although  the 
specimen  may  also  be  mounted  on  the  cover-glass  or  on  isinglass.  The  cover- 
glass  or  slide  or  isinglass,  as  the  case  may  be,  must  first  be  thoroughly 
cleansed.  If  new,  they  should  first  be  moistened  with  dilute  (20 — 30%)  alco- 
hol, and  dried  with  a  clean  cloth.  Then  a  minute  drop  of  egg  albumin  glyce- 
rine (cut  up  the  white  of  an  egg,  filter  and  mix  with  an  equal  part  of  glycerine 
and  a  large  crystal  of  thymol)  is  placed  on  the  centre  of  the  slide  and  spread 
thoroughly  with  the  tip  of  one  finger.  A  large  drop  of  water  is  quickly  placed 
on  this  layer  of  egg  albumin  and  allowed  to  spread  and  the  section  placed 
therein.  The  slide  is  now  held  over  a  flame,  the  heat  being  estimated  by  the 
hand,  until  the  sections  flatten  out  completely,  without  being  burned,  the  super- 
fluous water  is  then  decanted,  the  sections  arranged,  and  the  slide  placed  in  the 
upper  part  of  the  thermostat  to  dry.  A  few  hours  will  suffice,  after  which  the 
sections  will  be  found  to  adhere  completely. 

For  sections  which  are  not  to  be  stained  another  method  is  more  suited. 
The  slide  is  coated  with  a  thin  layer  of  a  mixture  of  oil  of  cloves  and  collodium 
(collodium  1  part,  oil  of  cloves  3 — 4  parts),  the  sections  are  pressed  down 


46 

with  a  brush  or  dry  finger-tip  and  can  then  be  transferred  to  xylol  to  dissolve 
the  paraffin. 

The  Celloidin  Section  Method 

Celloidin. 

Celloidin,  a  highly  concentrated  solution  of  guncotton,  dinitrocellulose, 
in  a  mixture  of  alcohol  and  ether,  is  sold  in  the  form  of  rectangular  plates  of 
a  finger's  thickness,  having  a  consistency  of  cartilage.  They  are  saturated 
with  alcohol  and  packed  in  hermetically  sealed  tin  boxes  to  avoid  drying. 

The  Preparation  of 
Hie    Celloidin    Solution. 

To  prepare  a  solution  suitable  for  embedding,  the  Celloidin  must  first  be 
carefully  dried.  The  plate  is  cut  into  strips,  which  are  in  turn  divided  into 
smaller  pieces  and  put  in  a  glass  beaker.  After  protecting  the  contents  from 
dust  by  covering  with  a  sheet  of  paper,  the  beaker  is  put  in  the  thermostat. 
After  two  to  three  days  the  celloidin  particles  will  have  dried  and  shrunk  to 
opaque  hornlike  chips.  Care  must  be  taken  in  the  following  steps,  as  the 
celloidin  in  this  dried  state  is  explosive.  The  chips  arc  put  in  a  glass-stoppered 
bottle  (of  800 — 1,000  cm3  volume),  which  must  be  absolutely  dry,  and  are 
covered  with  250  cm3  of  absolute  alcohol.  The  mixture  is  frequently  shaken 
or  stirred  with  a  glass  rod.  After  twenty-four  hours  the  chips  will  be  softened 
and  swelled  to  a  glassy  mass.  250  cm3  of  water-free  ether  are  added  and,  if 
promptly  stirred  and  shaken,  the  solution  will  be  complete  in  from  two  to  three 
days.  We  now  have  a  mass  of  honeylike  consistency,  the  celloidin  stock 
solution.  From  the  latter  we  prepare  two  dilutions :  Celloidin  I  ( 1  part 
of  stock  to  3  parts  of  alcohol-ether),  and  Celloidin  II  (1  part  of  stock  to  1 
part  of  alcohol-ether).  Both  solutions  should  be  kept  in  well-corked  bottles. 

Dehydration  and  Saturation. 

As  regards  celloidin  embedding  we  find  that  here,  too,  thorough  dehydration 
is  of  prime  importance ;  we  therefore  have  to  gradually  transfer  our  specimen 
into  stronger  and  finally  absolute  alcohol,  the  latter  being  changed  at  least 
once.  It  is  then  brought  into  a  mixture  of  equal  parts  of  absolute  alcohol  and 
water-free  ether,  and  from  there  into  Celloidin  I,  i.e.,  the  thin  celloidin  solu- 
tion. The  length  of  time  spent  in  the  latter  (of  course  in  a  well-closed  bottle) 
depends  entirely  on  the  size  of  the  specimen.  For  very  small  objects  two  days 
are  sufficient,  larger  ones  must  remain  a  correspondingly  longer  period  up  to 
two  or  three  weeks.  From  this  thin  solution  the  specimen  is  transferred  to  the 
thicker  Celloidin  II,  and  thence  to  the  stock  solution,  remaining  in  each  an 
equal  length  of  time. 

Preparation  and  Preservation 
of  the  Celloidin  BlocTc. 

After  saturation  with  the  stock  solution  the  specimen  with  the  retained 
celloidin  is  placed  on  a  correspondingly  large  dry  wooden  block,  exposed  to 
the  air  for  about  ten  minutes  and  preserved  in  a  large  quantity  of  70 — 80% 
alcohol.  After  being  acted  on  by  the  alcohol  for  twenty-four  hours  the  speci- 
men will  have  attained  a  consistency  adapted  for  cutting.  Before  using,  the 
wooden  blocks  must  be  treated  for  several  days  with  95%  alcohol,  preferably 


47 

warm,  to  free  them  from  tannic  acid  and  resin.  Stabilit,  a  material  often 
used  in  electro-technique,  may  be  used  instead  of  wood ;  it  can  easily  be  divided 
with  a  saw. 

Preparation   of 

the  Celloidin  Section. 

The  microtome,  previously  mentioned,  with  attached -clamp,  can  readily 
be  used  for  the  preparation  of  celloidin  sections.  The  knife  is  adjusted  as 
obliquely  as  possible,  forming  an  acute  angle  with  the  long  axis  of  the  micro- 
tome, so  that  in  cutting  the  entire  edge  is  made  use  of.  This  presupposes,  of 
course,  that  the  cutting  edge  is  immaculate.  During  the  cutting  the  knife  is 
constantly  moistened  with  70 — 80%  alcohol  by  means  of  a  large  camel's-hair 
brush,  so  that  the  sections  float  on  the  edge.  Tissue  forceps  will  serve  to 
transfer  the  sections  from  the  knife  to  the  alcohol  dish.  As  soiling  of  the 
microtome  with  alcohol  cannot  be  avoided  during  all  these  procedures,  a 
thorough  cleaning  of  the  instrument  afterward  is  essential. 


SELECTION  OF  THE   CUTTING  METHOD   IN  SPECIAL   CASES 

Each  of  the  methods  described  has  its  own  merits  as  well  as  disadvantages. 
The  freezing  method  excels  the  other  two  without  doubt,  on  account  of  its 
ready  execution  and  rapidity.  Here  our  specimen  does  not  come  in  contact 
with  any  extraneous  reagents,  excepting  the  formalin  solution,  and  therefore 
this  represents  the  most  conservative  method,  altering  the  staining  properties 
to  the  least  extent.  On  the  other  hand,  this  method  presents  a  great  disad- 
vantage, met  in  objects  in  which  the  component  parts  are  only  loosely  con- 
nected or  are  separated  by  interspaces,  where  this  connection  is  easily,  often 
unavoidably,  destroyed,  and  this  fault  will  not  be  remedied  as  long  as  we  fail 
to  discover  a  suitable  embedding  method  for  fresh  objects  or  those  kept  in 
water. 

The  paraffin  method  is  doubtless  the  most  aggressive  of  the  three  methods, 
necessitating  not  only  a  thorough  treatment  with  alcohol,  but  also  the  heat- 
ing of  the  specimen  to  a  considerable  degree.  The  advantage  in  this  procedure 
lies  in  the  fact  that  specimens  so  treated  allow  of  the  most  delicate  cuts,  and 
that  in  these  sections  the  original  position  of  the  parts  is  absolutely  preserved, 
and  furthermore  that  the  after-treatment  is  the  easiest  imaginable.  Its  present 
supremacy  in  our  histologic  and  zoologic  laboratories  is  due  to  these  ad- 
vantages. 

Midway  between  freezing  and  paraffin  methods  stands  the  celloidin  section 
method,  which  avoids  heating  the  specimen,  but  requires  a  longer  saturation 
and  the  most  careful  manipulation.  Thin  sections,  i.e.,  5 — 10  [*•,  may  be  ob- 
tained in  either  of  the  three  methods.  The  thinnest  are  undoubtedly  fur- 
nished by  the  paraffin  method,  at  least  when  the  specimen  is  small.  When  deal- 
ing with  larger  objects  the  freezing  method  will  answer  as  well  or  often  better. 

The  reader  can  deduct  from  these  suggestions  which  method  be  best  suited 
for  a  particular  case.  We  always  give  the  freezing  method  the  first  rank,  as 
being  the  simplest  and  least  aggressive,  and  choose  the  others  only  when  sec- 


48 

tions  of  the  finest  precision  are  wanted,  or  when  the  structure  of  the  tissue 
calls  for  embedding. 


APPENDIX  TO  THE   PREPARATION   OF   SECTIONS 

DECALCIFICATION 

The  hard  structures  of  the  human  body,  viz.,  bones  and  teeth,  due  to  de- 
posits of  lime  salts,  possess  such  firmness  that  they  resist  cutting.  To  render 
them  soft,  we  must  aim  to  remove  these  salts  by  treating  them  with  their 
solvents,  viz.,  acids.-  This  process  has  received  the  name  of  decalcification. 

In  many  cases  the  fixing  solution  can  be  used  as  such  by  protracting  its 
application ;  e.g.,  chrome-osmium-acetic  acid  and  sublimate-nitric  acid  are 
excellent  dccalcifiers  and  yield  good  results  if  small  specimens  are  suspended 
in  large  amounts  of  the  liquid  and  the  latter  is  changed  frequently. 

As  a  general  rule,  however,  we  recommend  to  fix  the  object  first  thoroughly 
and  then  expose  it  to  the  action  of  a  decalcifier.  As  such  only  those  acids  can 
be  considered  which  can  change  the  carbonates  or  phosphates  of  the  hard 
structure  in  question  into  their  respective  soluble  salt. 

Among  these  acids  nitric  again  ranks  foremost.  As  a  decalcifier  we  use  it 
in  a  5%  watery  solution,  immersing  the  fixed  and  washed  specimens  in  large 
quantities  of  same.  To  expedite  decalcification,  the  specimen  is  frequently 
shaken,  or  it  may  be  suspended  in  the  upper  layers  of  the  fluid  and  the  latter 
changed  repeatedly.  The  duration  of  this  process  of  course  depends  on  the 
size  of  the  object  and  the  amount  of  lime  contained  in  it.  In  order  to  test  the 
progress  of  decalcification  the  specimen  may  be  pierced  with  a  needle  or  an 
attempt  at  cutting  with  the  razor  may  be  made,  unless  such  procedures  are 
contraindicated  for  other  reasons.  With  fair  sized  pieces  of  only  moderately 
old  bone  two  to  four  days  will  usually  complete  the  process,  for  whole  teeth 
eight  to  ten  days  will  suffice.  After  decalcification  the  acid  must  not  be 
directly  washed  out  with  water,  but  the  specimen  first  immersed  in  10%  forma- 
lin (which  is  changed  frequently)  or  in  5%  Glauber  salt  solution  (also  to  be 
changed  repeatedly)  for  twenty-four  hours,  after  which  the  washing  can  be 
done  without  any  harm  resulting. 

Of  the  other  decalcifiers  we  will  only  mention  the  trichloracetic  acid,  the 
method  of  procedure  being  identical  with  that  of  nitric  acid. 


STAINING  METHODS 

Staining. 

By  staining  we  mean  that  process  by  which  an  unstained  body,  treated 
with  the  solution  of  a  stained  body,  appears  stained  itself.  The  color  result- 
ing will  usually  be  the  same  as  that  of  the  solution  used;  however,  under  certain 
circumstances  this  may  not  be  the  case.  Dyeing  is  a  primeval  art,  owing  its 
development  to  the  beauty  sense  of  mankind.  The  methods  employed  for  the 
staining  of  our  microscopic  specimens  are  largely  drawn  from  the  practical 
dyeing  process,  but  not  with  a  view  as  to  beauty  in  colors,  but  solely  because 


49 

the  staining  is  an  important,  if  not  the  most  important,  aid  in  the  diagnosis 

of  tissues  and  their  component  parts. 

% 

Stains. 

All  stains  used  in  microtechnique  are  organic  compounds,  viz.,  they  contain 
carbon  and  hydrogen.  They  either  owe  their  origin  directly  to  the  animal  or 
vegetable  kingdom,  or,  as  in  the  majority  of  cases,  they  are  artificially  pre- 
pared (synthetic),  so  that  we  must  differentiate  between  natural  and  arti~ 
fidal  stains. 

The  Essentiality 
of  Staining. 

It  has  not  been  decided  as  yet  with  accuracy  what  the  process  is  that 
causes  an  organic  body  to  appear  colored,  i.e.,  to  let  certain  rays  of  the  spec- 
trum pass  through,  and  reflect  others,  and  absorb  still  others,  but  so  much 
we  may  safely  assume,  that  this  property  is  founded  in  the  chemical  constitu- 
tion of  the  body,  and  a  number  of  atom  groups  are  already  known  which  pos- 
sess the  capability,  when  entering  an  unstained  body,  to  change  the  same  into 
a  dye.  In  the  same  manner  the  essentials  of  staining  have  remained  rather 
obscure.  Three  theories  have  so  far  been  expounded.  The  mechanical 
theory  assumes  that  physical  powers  alone  are  concerned  in  driving  the 
staining  solution  into  the  tissues,  namely,  osmosis  and  capillary  attraction. 
Once  in  the  tissues  the  dye  is  condensed  by  absorption  in  a  similar  manner,  as 
animal  charcoal  may  detract  all  the  dye  out  of  a  stain  solution  and  render 
the  latter  colorless.  The  chemical  theory,  on  the  contrary,  claims  that 
chemical,  saltlike  compounds  are  formed  during  the  process  of  staining  be- 
tween the  stain,  respectively,  its  components  and  the  albuminous  substances  of 
our  specimen.  As  we  have  discussed  previously,  these  albuminous  bodies  partly 
take  on  the  character  of  acids,  partly  that  of  bases,  and  it  therefore  seems 
quite  reasonable  that  by  their  combination  with  the  stain  acids,  respectively, 
stain  bases  of  our  dyes,  saltlike  compounds  result.  The  third  theory  finally  con- 
siders the  stained  preparation  as  a  solidified  solution  of'the  stain  in  the  tissues. 
This  is  not  the  place  to  enter  into  a  discussion  of  the«merits  of  each  of  these 
theories.  Suffice  it  to  say  that  without  doubt  chemical  processes  are  con- 
cerned in  the  phenomenon  of  staining,  which,  however,  do  not  exclude  the  pos- 
sibility of  processes  of  a  physical  nature  occurring  at  the  same  time. 

Preparation  of  the 
Color  Bath. 

A  stain  to  be  of  use  in  our  work  must  be  soluble.  Solvents  are  primarily 
water,  but  also  alcohol,  rarely  glycerine ;  furthermore,  salt  solutions,  weak 
acids  and  alkalies  are  all  used.  To  increase  the  staining  power  of  some  of  the 
watery  solutions  of  dye,  aniline  is  added,  or  instead  of  dissolving  the  coloring 
matter  in  water,  we  use  aniline  water,  which  can  be  prepared  by  mixing  a 
small  amount  of  aniline  with  distilled  water,  thoroughly  shaking  it  and  filtering 
the  opaque  mixture  through  a  previously  moistened  filter. 

Concentration    of   the   Stain. 

As  a  rule,  we  prepare  staining  solutions  of  a  fixed  strength,  i.e.,  a  certain 
amount  of  dye  is  dissolved  in  a  given  amount  of  solvent,  so  that  all  of  the  dye 


50 

will  go  into  solution.  The  dissolving  takes  place  either  at  room  temperature 
or  the  solvent  is  previously  heated.  Often,  however,  we  use  the  so-called  con- 
centrated solution,  which  is  made  by  introducing  an  excess  of  the  dye  into  the 
solvent  and  shaking  repeatedly ;  after  a  while  we  find  above  the  sediment  of 
the  excess  a  solution  of  maximum  concentration.  Owing  to  the  fact  that  in 
most  cases  the  dyes  are  more  highly  soluble  at  a  higher  temperature  than  at 
some  lower,  the  strength  of  such  solutions  must  needs  fluctuate  with  the  tem- 
perature in  which  they  are  kept.  When  using  concentrated  solutions  the  de- 
canting must  be  done  with  great  care,  to  avoid  picking  up  some  of  the  small 
solid  particles  of  dye  from  below. 

It  is  difficult  to  say  in  general  which  concentration  would  be  apt  to  yield 
the  best  results.  We  often  hear  it  mentioned  that  thin  solutions  work  better 
than  the  concentrated.  That  may  be  the  case  with  some  certain  dyes,  but  can- 
not be  adopted  as  a  general  principle.  In  short,  the  concentration  must  de- 
pend upon  the  nature  of  the  dye  and  the  particular  case  in  which  it  is  to  be 
used. 

Temperature  of  the 
Staining  Bath. 

As  a  rule,  we  stain  at  room  temperature,  but  special  circumstances  may 
demand  a  higher  temperature,  when  the  staining  solution  must  be  warmed ;  this 
is  identical  with  the  custom  of  heating  the  solutions  in  practical  dyeing.  Beyond 
doubt  the  heated  dye  will  penetrate  tissues  in  a  shorter  period  of  time  and 
the  staining  will  be  more  intensive  than  if  lower  temperatures  prevail. 

Staining  of  the 
Living  Specimen. 

Living  specimens,  as  well  as  preserved,  may  be  stained.  The  former  is 
called  vital  staining  or  staining  in  the  living  state.  A  vital  staining, 
in  the  strict  sense  of  the  word,  can  only  be  executed  in  the  living  animal  body 
itself,  by  incorporating  into  the  latter  the  desired  dye  in  some  appropriate 
fashion.  Vital  staining  is  of  great  importance  in  research,  and,  although  still 
in  its  infancy  of  elaboration,  the  results  obtained  have  been  brilliant.  In  the 
higher  animal  the  incorporation  of  dye  may  be  accomplished  by  means  of 
feeding,  the  dye  being  mixed  with  the  food.  The  dye  having  been  dissolved 
in  the  chyme,  absorption  takes  place  in  the  intestines.  A  quicker  method  is  to 
inject  the  watery  solution  hypodermatically,  and  finally  the  intravenous 
method  may  be  resorted  to,  by  introducing  the  stain  into  a  vein  of  the 
anaesthetized  animal.  Of  course  the  dye  must  not  be  poisonous  enough  to  cause 
a  cessation  of  heart  action.  If  such  should  be  the  case,  the  animal  is  allowed 
to  bleed  to  death,  after  which  the  circulatory  system  is  flooded  under  pressure, 
the  place  of  introduction  being  the  heart  or  an  artery.  The  latter  method  can 
be  used  to  good  advantage  in  the  human  body,  e.g.,  an  amputated  extremity. 

Staining  of  the 
Surviving  Specimen. 

We  often  speak  of  vital  staining,  when  small  sections  are  taken  from  the 
living  or  recently  killed  animal  and  subjected  to  the  action  of  a  staining 
fluid.  It  is  a  question  whether  we  have  here  really  vital  staining,  since  there 
are  no  criteria  which  would  show  us  that  such  a  section  of  tissue  or  its  com- 


51 

ponents  at  the  moment  of  staining  are  still  living  or  have  died,  a  question 
which  might  also  enter  into  the  vital  staining  proper.  Hcnce_it  might  be  wiser 
to  designate  such  procedures  as  staining  of  fresh  specimens,  i.e.,  such  as  are 
not  preserved.  Weak  solutions  should  be  used  for  this  purpose  and,  if  possible, 
the  dye  should  be  dissolved  in  some  indifferent  fluid,  e.g.,  normal  saline  or 
Ringer's  fluid.  We  can  either  take  small  particles  of  the  organ  with  scissors  or 
razor,  or  we  can  first  make  frozen  sections  of  these  particles  and  stain  the 
latter.  The  chopping  method  is  also  useful,  the  specimens  being  quickly 
chopped  on  the  slide,  after  which  the  stain  is  added,  the  specimen  put  in  a 
moist  chamber  and  the  whole  eventually  placed  in  the  thermostat  at  a  tem- 
perature of  38 — 40°. 

Staining  of  the 

Preserved  Specimen. 

The  staining  of  preserved  objects  is  far  more  common.  Here  we  must 
always  bear  in  mind  that  our  fixation  and  preserving  process  causes  invariably 
radical  changes  in  the  chemical  composition  of  the  albumins,  which  make  up 
cells  and  tissues.  One  should  therefore  "be  guarded  in  promising  results,  which 
are  to  be  obtained  with  these  methods. 

Lump  and  Section  Staining. 

Two  separate  methods  of  procedure  are  to  be  distinguished  in  the  staining 
of  preserved  objects;  on  the  one  hand  we  may  stain  larger  or  smaller  lumps 
of  tissue  in  toto,  on  the  other  we  subject  the  sections  made  of  the  piece  of 
tissue  to  the  dye.  The  lump  staining  requires  a  dye  of  great  power  of  diffu- 
sion, unless  we  deal  with  membranous  structures.  Most  of  the  watery  solutions 
lack  this  quality,  whence  it  is  necessary,  with  few  exceptions,  to  resort  to  alco- 
holic solutions  when  staining  in  lump  is  desirable.  Another  disadvantage  of 
lump  staining  lies  in  the  fact  that  we  are  not  able  to  control  the  staining  pro- 
cess sufficiently.  For  these  reasons  this  method  is  used  only  seldom  in  his- 
tology, while  embryology  employs  it  frequently  to  this  date.  In  section  stain- 
ing there  is  no  difficulty  in  controlling  the  progress  of  the  staining  process  and 
to  interrupt  it  at  the  proper  time,  and  therefore  it  has  been  adopted  as  the 
superior  of  the  two  methods. 

Staining  of  Frozen  Sections. 

For  the  staining  of  frozen  sections  watch-glasses  of  different  sizes  with 
ground  edges,  convex  on  the  surface  and  straight  on  the  bottom,  on  which  they 
rest,  are  most  serviceable.  As  a  cover  a  glass  plate  or  a  similar  watch-glass 
of  the  same  size  may  be  used.  The  sections  are  placed  into  the  staining  fluid 
by  means  of  a  curved,  well-polished  needle  or,  better  still,  with  slightly  curved 
glass  needles,  which  can  be  easily  made  from  a  glass  rod  or  a  thick-walled 
glass  tube  (barometer  tube)  in  any  desired  size.  It  is  important  that  these 
needles  be  smoothly  polished ;  if  they  lack  in  even  smoothness  or  present  rough- 
ness in  the  least  degree,  sections  may  easily  adhere  and  tear.  T|ie  specimens 
are  removed  in  the  same  manner.  When  the  stain  is  opaque,  difficulty  in  re- 
moving the  sections  may  be  encountered.  When  staining  but  a  few'  sections, 
only  a  few  drops  of  the  solution  are  necessary ;  when  staining  a  larger  amount 


52 

in  a  correspondingly  larger  quantity  of  dye,  the  curved  needle  is  guided  along 
the  bottom  of  the  dish  and  the  sections  are  fished  out  gradually. 

Staining  of  Paraffin  " 
Sections  on  the  Slide. 

The  staining  of  paraffin  sections  previously  pasted  on  the  slide  is  best 
performed  in  cylindrical  glasses,  which  must  be  slightly  higher  and  a  trifle 
broader  than  our  slides.  The  first  aim  will  be  to  remove  the  paraffin  from  the 
section,  since  its  presence  renders  staining  difficult,  although  not  impossible. 
A  good  solvent  is  found  in  xylol,  C0  H4  (CH3)2,  a  waterlike  transparent  fluid, 
lighter  than  water  and  unable  to  mix  with  water  in  any  proportion.  The 
paraffin  will  dissolve  in  a  few  seconds,  if  the  slide  is  lightly  agitated  to  and  fro, 
and  if  the  sections  are  not  too  thick.  In  order  to  be  able  to  transfer  the  speci- 
men into  our  watery  staining  solution,  we  must  now  remove  all  the  xylol  from 
it,  which  is  accomplished  by  the  use  of  absolute  alcohol;  from  the  latter 
the  sections  are  first  transferred  to  a  more  dilute  grade  of  alcohol,  say  90%, 
before  the  staining  proper  can  begin.  First  the  watery  stain  will  not  penetrate 
the  section,  but  after  moving  the  latter  to  and  fro  a  few  times,  it  will  enter  the 
tissue.  Practically  it  is  best  to  have  several  of  these  glasses  side  by  side,  the 
first  containing  xylol,  the  second  absolute  alcohol,  the  third  90%  alcohol,  and 
the  fourth  the  staining  fluid.  It  is  of  advantage  to  insert  a  fifth  glass  between 
the  third  and  fourth,  containing  water.  Specimens  are  transferred  from  one 
fluid  to  the  other,  several  slides  being  placed  in  one  glass  if  necessary,  by  plac- 
ing them  in  pairs  with  their  backs  approximated.  The  film  sides  of  course 
must  not  be  touched. in  any  way  in  order  to  avoid  laceration  of  the  section. 

Staining  cf  Cclloidin  Sections. 

Celloidin  Sections  are  treated  in  a  similar  manner  as  are  frozen  sec- 
tions, with  this  exception,  that  less  care  need  be  taken  in  transferring.  The 
specimen  is  simply  put  from  alcohol  to  water  and  thence  into  the  stain.  If 
the  latter  is  an  alcoholic  solution,  the  water,  of  course,  is  omitted. 

Simple  and 
Multiple  Staining. 

Depending  upon  whether  we  stain  with  one  or  with  several  dyes  we 
differentiate  between  simple  and  multiple  staining.  In  multiple  staining 
we  naturally  select  dyestuffs  which  differ  widely,  in  general,  contrast-stains. 
When  several  stains  are  used  in  succession  we  speak  of  successive  mul- 
tiple staining;  again,  when  a  mixture  of  several  dyes  is  used  for  staining 
we  call  the  process  simultaneous  multiple  staining  or  compound 
staining. 

Progressive  and 
Regressive  Staining. 

If  the  staining  process  is  interrupted  at  its  height,  i.e.,  the  optimum  of 
the  process,  we  speak  of  progressive  staining;  in  that  case  we  need  only 
wash  out  the  excess  staining  solution  from  the  specimen.  When  using  the 
regressive  Staining  method,  we  overstain  the  section  intentionally,  bringing 
it  to  the  maximum  of  the  process,  the  optimum  being  gained  by  treating  the 


53 

specimen  with  a  solvent  of  the  dye  used,  or  by  partly  destroying  the  stain. 
Such  agents  are  known  as  differentiation  agents. 

Substantive  and 
Adjective  Staining. 

According  to  practical  dyeing  we  can  also  differentiate  between  substan- 
tive and  adjective  method.  The  process  is  substantive  when  a  simple  solu- 
tion of  a  dye  is  used  without  any  adjunct  agent.  In  the  adjective  method 
we  have  besides  dye  and  specimen  a  third  factor,  without  which  the  dye  will 
fail  to  stain  the  tissue.  Such  agents  are  designated  as  mordants  or  bases, 
and  we  speak  of  a  mordant  dye,  when  it  alone  has  little  or  no  staining  power, 
the  latter  only  being  developed  when  the  base  is  added.  Here,  too,  chemistry 
has  given  us  the  key  to  the  relation  existing  between  the  composition  of  a  dye 
and  its  staining  power,  inasmuch  as  we  know  that  all  dyestuffs  containing  two 
hydroxyl  groups  in  ortho  position  are  mordant  dyes. 

Mordants. 

Agents  of  all  sorts  of  chemical  structures  are  used  as  bases  in  commercial 
dyeing,  e.g.,  acids,  oils  and  the  product  of  the  action  of  the  former  on  the 
latter,  tannins,  metal  oxides  and  others.  In  microtechnique  we  use  almost  ex- 
clusively metal  oxides  for  this  purpose.  The  mordant1  dyes  possess  the  prop- 
erty of  forming  with  metal  oxides  saltlike  compounds  of  high  staining 
power,  being  soluble  in  water  only  to  a  small  degree.  Such  compounds  are 
designated  as  color  lakes.  We  can  prepare  the  latter  in  vitro,  put  them  into 
solution  in  the  proper  manner,  and  then  stain  with  this  solution  of  the  lake ; 
i.e.,  simultaneous  mordant  dyeing,  or  we  first  let  a  solution  of  our  base 
act  on  the  specimen  and  stain  it  thereafter,  succedaneous  mordant  dye- 
ing. Then  the  lake  is  created  in  the  specimen.  Mordant  staining  plays  an 
important  role  in  microtechnique,  the  simultaneous  process  as  well  as  the  suc- 
cedaneous. 

Diffuse  and  Elective  Staining. 

The  result  of  a  staining  process  may  be  diffuse  or  elective.  It  will  be 
diffuse  if  all  the  component  parts  of  the  specimen  appear  equally  stained. 
Staining  is  called  elective  when  either  certain  parts  of  a  tissue  are  affected, 
while  others  remain  unstained,  or  when  all  the  parts  are  stained  but  differ  in 
shade  of  the  same  color  or  under  the  most  favorable  circumstances  appear  ac- 
tually in  different  colors.  It  is  evident  at  once  that  diffuse  staining  is  of  small 
value,  and  that  our  aim  must  be  to  accomplish  elective  staining.  This  end  may 
be  accomplished  in  various  manners,  since  experience  has  taught  us  that  the 
different  dyes  have  a  different  affinity  to  certain  kinds  of  tissue,  so  that,  when 
treated  progressively,  they  are  stained  more  quickly,  while  under  the  regressive 
method  they  retain  the  particular  dye  correspondingly  longer. 

A  specimen  treated  with  the  simultaneous  or  succedaneous  method  will  show 
that,  under  proper  technique,  certain  dyes  are  taken  up  by  certain  parts  of 
tissues ;  e.g.,  certain  dyes  only  stain  the  nuclear  chromatin,  the  basic  sub- 

1The  terms,  "mordant"  and  "base,"  are  used  interchangeably  to  represent  the  Ger- 
man "Beize,"  the  word  mordant  being  of  French  origin:  mordre  =  to  etch. — (The 
Translator.) 


54 

stance  of  cartilage,  mucus,  certain  kinds  of  cellular  granulations ;  others, 
again,  will  affect  the  nucleoli,  the  protoplasm  of  the  cell  body,  the  haemoglobin 
of  the  red  blood  corpuscles,  the  connective  tissue  shreds,  the  basic  substance  of 
bone,  etc.  We  have  thus  found  that  the  group  mentioned  first  is  stained  by 
basic  dyes,  i.e.,  combinations  of  color  bases  with  any  desired  acid,  while  the 
latter  is  affected  by  add  dyes,  i.e.,  color  acids  or  their  alkaline  salts ;  we 
may  therefore  call  the  first  group  of  tissues  or  parts  thereof  basophilic,  the 
latter  acidophilic. 

Metachromatic  Staining. 

It  is  not  always  necessary  to  treat  a  specimen  with  two  different  colors, 
say  red  and  blue,  in  order  to  obtain  red  in  certain  tissue  elements  and  blue  in 
others ;  we  have  certain  chemically  uniform  dyes,  which  possess  the  property 
of  staining  in  two  different  colors,  or  even  will  bring  out  different  shades  of 
these  colors  in  various  tissue  components.  Such  dyes  are  known  as  meta- 
chromatic.  Metachromasia  is  a  virtue  possessed  by  many,  but  principally 
by  basic  dyes,  where  the  free  base  has  a  different  color  than  the  salt  repre- 
sented by  the  dye;  e.g.,  not  infrequently  we  find  that  a  color  base  is  staining 
red,  while  its  hydrochloric  acid  salt  will  give  a  blue  color.  The  latter  we  use 
as  a  dye,  since  the  color  bases  are,  as  a  rule,  insoluble  in  water.  If  we  now  pro- 
ceed to  stain  in  the  watery  solution  of  such  a  blue  dye,  certain  elements  will  be 
stained  orthochromatic,  i.e.,  in  the  color  of  the  solution,  viz.,  blue,  others  meta- 
chromatic,  in  the  color  of  the  base,  viz.,  red.  Metachromatic  staining  will 
produce  the  greatest  contrasts  on  the  fresh,  unprepared  tissue  and  is  greatly 
influenced  by  our  various  fixing  methods.  A  dye,  in  order  to  develop  meta- 
chromatic  properties,  must  generally  be  in  watery  solution,  since  alcoholic 
solutions  show  little  or  no  metachromasia,  the  latter  also  being  usually  de- 
stroyed if  the  specimen  is  afterward  treated  with  alcohol.  A  strong  meta- 
chromasia and  a  decided  basophilia  may  in  many  tissue  elements  go  hand  in 
hand,  e.g.,  mucus,  cartilaginous  substance,  certain  cell  granulations ;  on  the 
contrary  the  nuclear  chromatin  is  decidedly  basophilic,  but  shows  less  meta- 
chromasia as  does  the  greatly  acidophilic  collagenous  connective  tissue.  The 
cell  protoplasm  is  always  orthochromatic.  Definite  knowledge  of  the  cause 
of  metachromasia  is  up  to  the  present  not  claimed ;  perhaps  the  phenome- 
non is  due  to  a  tautomeric  combination  of  the  color  base  in  question  with  the 
acid. 

After  this  preliminary  discussion  of  dyes  and  staining  we  will  proceed  to 
study  singly  the  most  important  stains. 


STAINS   OF   ANIMAL   ORIGIN 

Cochenille  (Cocheneal) 
Carmine. 

The  mother  substance  of  carmine  is  the  cochenille.  The  latter  is  a  name 
given  to  the  dried  female  of  the  Coccus  Cacti,  which  habitates  several  species 
of  the  Opuntia  Cacti  in  Mexico  and  Central  America.  The  female  insects  are 
gathered  from  the  plants  shortly  before  shedding  the  eggs,  they  are  then  killed 


55 

in  hot  water  and  dried  in  the  oven.     Approximately  140,000  animals  will  yield 
1  kg.1  of  cochenille. 

Carmine. 

Carmine  is  manufactured  from  the  cochenille  by  extraction  with  alum  and 
tartar  and  occurs  as  a  deeply  red,  earthy,  friable  mass,  being  put  into  com- 
merce in  smaller-or  larger  lumps.  It  is  totally  insoluble  in  water  and  in  alcohol, 
soluble  in  alkalies,  ammonia,  acids,  borax,  and  alum  solutions. 

Carminic  Acid. 

Chemically  we  must  consider  carmine  as  a  combination  of  clay-lime- 
albumin  with  carminic  acid.  The  latter  is  the  coloring  principle  of  carmine 
and  in  its  pure  state  forms  a  beautiful  red  crystalline  powder,  readily  soluble 
in  water  and  alcohol.  With  alkalies  it  forms  soluble,  with  earthy  alkalies  and 
the  heavy  metals  insoluble  salts. 

In  microtechnique  the  staining  solutions  are  prepared  from  either  cochi- 
nille  or  carmine,  or  carminic  acid,  as  well.  The  best  and  most  constant  results 
are  without  doubt  obtained  from  solutions  made  of  chemically  pure  carminic 
acid,  and  for  this  reason  we  shall  use  such  solutions  almost  exclusively. 

Carmalurn. 

Make  a  hot  solution  of  5%  potash  alum,  cool  and  filter;  heat  the  filtrate 
and  dissolve  in  it  carminic  acid  to  the  extent  of  0.5%.  A  deep  bluish  red  solu- 
tion results,  which  after  cooling  is  filtered  and  preserved  in  0.5 — 1%  formalin, 
this  solution  will  stain  sections  very  rapidly  and  intensively,  imparting  a  bluish 
red  tone.  After  staining  for  usually  ten  to  fifteen  minutes  the  sections  are 
placed  in  a  5%  alum  solution  for  a  few  seconds  and  then  washed  in  water.  In 
this  way  an  almost  pure  staining  of  the  nucleus  is  obtained.  This  stain  is  also 
adaptable  to  block  staining.  The  blocks  must  not  be  too  large  and  should  re- 
main in  the  staining  fluid  from  twenty-four  to  forty-eight  hours,  after  which 
they  are  washed  in  water. 

Paracarmine. 

This  preparation  is  of  still  greater  intensity  and  can  be  made  by  dissolv- 
ing 1  gm  of  carminic  acid,  0.5  gm  of  chloraluminum'^and  4  gms  of  chlorcal- 
cium  2  in  100  cm3  of  70%  alcohol,  heating  carefully.  When  the  precipitate  has 
'settled,  filter  and  keep  in  a  well-corked  bottle.  For  staining,  this  solution  is 
diluted  with  five  to  ten  times  its  volume  of  70%  alcohol,  and  acetic  acid  is 
added  to  the  small  extent  of  1 — 2  drops  to  each.  Paracarmine  stains  more 
rapidly  and  intensively  than  carmalum  and  can  be  used  to  the  same  advantage 
for  sections  or  blocks.  Alcohol,  70%,  is  used  for  washing;  2%  acetic  acid  can 
be  added  to  the  alcohol,  if  an  absolute  pure  staining  of  the  nucleus  is  desired. 


STAINS    OF    VEGETABLE     ORIGIN 

Flora  furnishes  us  with  dyes  of  great  variety  and  number,  which  formerly 
were  of  decided  importance  to  the  practical  dyer  and  are  even  to  this  day  used 

1Two  pounds.  2  Chlorides. 


56 

frequently  by  him.     For  our  purpose  only  two  deserve  mention:  haematoxylin 
and  indigo. 

Hcematoxylin. 

It  is  an  extract  made  from  the  wood  of  the  haematoxylon  campechianum,  a 
cassalpiniacea  found  in  Central  America,  which  is  extracted  in  the  form  of 
small  rhomboid,  colorless  crystals.  It  is  not  very  soluble  in-  cold  water,  but 
readily  so  in  hot  water,  alcohol  and  ether.  If  a  watery  or  alcoholic  solution  of 
haematoxylin  is  exposed  to  light  and  air,  it  will  first  assume  a  yellow  tinge ; 
later  it  will  become  brown. 

Hcematein. 

The  cause  of  this  change  in  color  is  found  in  the  fact  that  haematoxylin  is 
wholly  or  partly  oxidized  to  hasmatein.  The  latter  is  on  the  market  in  the 
form  of  a  brown  powder,  which  is  soluble  in  water,  alcohol,  ether,  glycerine, 
alkalies  and  ammonia  in  particular. 

Haematoxylin  per  se,  substantively,  cannot  be  used  as  a  stain ;  it  is  a  typical 
representative  of  a  mordant  dye,  forming,  with  most  metal  oxides,  strongly 
staining  lakes,  their  hue  varying  between  blue  and  black.  During  the  lake  for- 
mation the  haematoxylin  is  either  at  once  or  gradually  oxidized  to  haematein, 
and  the  more  advanced  the  oxidation  is,  the  better  the  lake  will  stain.  There 
Is,  of  course,  an  optimum  of  staining  power,  which  may  and  often  is  trans- 
gressed, since  the  oxidizing  process  does  not  stop  with  the  formation  of  hasma- 
tein, but  will  finally  lead  to  the  formation  of  oxalic  acid.  Haematoxylin  lakes 
«can  be  applied  in  either  the  simultaneous  or  the  succedaneous  method,  i.e.,  we 
can  stain  with  the  ready  solution  of  the  lake  or  we  may  produce  the  lake  in 
our  specimen  by  successive  applications  of  the  mordant  and  the  haematoxylin 
solution.  To  make  the  solution  we  may  use  either  haematoxylin  or  haematein. 

Hoemalum. 

This  is  a  clsj  haematein  lake.  Fifty  grams  of  alum  are  dissolved  in  1  litre 
of  hot  water,  and  the  solution  is  cooled  and  filtered.  One  gram  of  hasmatein  is 
dissolved  in  50  cm3  of  95%  alcohol  under  gentle  heating,  and  both  these  solu- 
tions are  mixed.  A  dark  bluish  violet  color  reaction  takes  place  at  once.  A 
sediment  is  common  and  renders  it  advisable  to  filter  before  using  the  stain. 
Haemalum  will  stain  sections  in  a  very  few  seconds;  the  specimen  is  washed  in- 
water,  which  will  impart  to  it  a  dark  blue  color.  If  overstained,  a  specimen 
can  be  reduced  by  being  placed  in  slightly  acidified  water  (2  drops  of  muriatic 
acid  to  50  cm3  of  water).  The  latter  solution  extracts  any  superfluous  dye, 
causing  at  the  same  time  a  change  in  color  from  the  blue  to  a  red  tint.  After 
being  thus  reduced  the  specimen  is  washed  until  it  again  assumes  a  blue  color. 

Iron  Hcematoxylin 

probably  furnishes  a  still  stronger  nuclear  color  result  than  the  preceding 
stain ;  it  is  prepared  as  follows :  10  gms  ammonium  ferrosulphate,  iron-alum, 
so-called,  are  dissolved  in  150  cm3  of  hot  water;  in  the  same  manner  1.6  gm 
haematoxylin  are  added  to  75  cm3  of  hot  water.  Both  solutions  are  cooled, 
one  is  poured  into  the  other  and  the  mixture  is  carefully  heated  over  a  small 
flame,  stirring  gently  until  the  seething  point  is  well  reached.  When  cooled, 


57 

the  solution,  now  dark  brown,  is  ready  for  use.  It  stains  very  rapidly  and' 
thoroughly.  If  overstating  has  taken  place  reduction  4s  Induced  in  the  same 
manner  as  in  haemalum. 

Hcematoxylin-Iron-A  lum. 

Two  solutions  are  used  for  this  succedaneous  mordant  staining,  which  is 
generally  spoken  of  as  Heidenhain  staining,  after  its  discoverer:  a  2.5%  solu- 
tion of  ammonium  ferro-sulphate  (iron- alum)  and  a  1%  solution  of  hsematoxy- 
lin,  which  latter  should  be  of  some  time  standing  and  brown.  The  sections  are 
first  placed  in  the  mordant  for  at  least  two  hours  or  more,  if  necessary,  after 
which  they  are  washed  for  a  short  time  and  transferred  into  the  color  solution, 
where  they  remain  overnight.  At  first  they  take  on  a  gray  color,  later  a  gray- 
ish blue,  then  deep  blue  and  finally  they  become  entirely  black.  If  reduction 
is  necessary  they  should,  after  a  brief  wash,  be  replaced  into  the  mordant, 
which  gradually  renders  them  lighter,  with  the  appearance  of  black  color  clouds 
in  the  solution.  This  reduction  or  differentiation  must  be  closely  watched  and 
should  be  controlled  under  the  microscope.  The  nucleoli  and  the  chromatin 
of  the  nuclei  resist  the  extraction  the  longest,  hence  this  procedure  is  a  means 
of  presenting  these  elements  absolutely  distinct.  After  the  desired  effect  is 
obtained  we  transfer  to  a  large  vessel  of  hydrant  water  for  at  least  ten 
minutes,  changing  the  water  repeatedly. 

H cemaioxylin-P 'otassium 
Dicliromate, 

a  lake  of  haematoxylin,  is  used  to  differentiate  the  medullated  fibres  within  the 
central  nervous  system,  pieces  of  which  are  first  saturated  with  the  chrome 
salt,  sections  made  thereof,  and  in  these  sections  the  lake  is  produced  by 
introducing  them  into  a  hasmatoxylin  solution.  For  further  details  of  the 
method  we  refer  the  reader  to  the  special  part  under  Nervous  System. 

Hcemaioxylin-Ph  ospho- 
Molybd  c  Acid. 

Dissolve  1.75  gm  of  hasmatoxylin  in  200  cm3  of  hot  water,  and  add 
10  cm3  of  a  10%  solution  of  phospho-molybdic  acid  and  -5  cm3  of  phenol 
(carbol.  acid  cryst.),  previously  liquefied  by  heating.  Sections,  previously 
treated  with  10%  phospho-molybdic  acid  for  ten  minutes  and  briefly  washed  in 
water,  are  kept  in  this  dark  blue  solution  for  from  fifteen  to  twenty  minutes, 
washed  in  water  and  transferred  to  alcohol. 

The  entire  specimen  appears  soft  grayish  blue,  not  very  distinctly  stained, 
but  from  this  background  the  collagenous  fibres  contrast  widely  in  their  deep 
blue  stain.  By  this  method  the  connective  tissue  fibres  are  distinctly  stained 
even  in  such  places  which  are  hard  to  bring  out  with  ordinary  stains. 

Indigo. 

A  dyestuff  known  in  the  most  ancient  times,  is  found  widely  distributed  in 
the  floral  kingdom,  but  its  chief  source  is  the  Indigofera,  a  papilionacea  culti- 
vated in  Bengal.  We  have  here  a  very  light,  deep  blue,  friable  mass,  insoluble 
in  water,  alcohol  and  ether,  but  soluble  in  aniline,  phenol  and  acetic  acid.  Con- 
centrated sulphuric  acid  will  dissolve  it,  with  the  formation  of  indigo-sulpho- 
acids.  When  treated  with  any  reducing  agent,  indigo  will  be  changed  to  indig- 


58 

white,  which  is   soluble  in  water.      The   formation  of  indig-white  and  indig- 
sulpho-acid  is  responsible  for  the  extensive  use  of  indigo  in  commercial  dyeing. 

Indig  carmine. 

In  microtechnique  we  use  a  preparation  produced  by  sulphuric  acid  acting 
as  indigo,  indig-carmine,  the  sodium  salt  of  indig-disulpho-acid,  occurring  as  a 
deep  blue  powder,  quite  readily  soluble  in  water,  slightly  or  not  soluble  in 
alcohol.  This  preparation  in  a  0.25%  solution  makes  an  excellent  plasma 
stain. 

ARTIFICIAL   STAINS 

Artificial  Stains. 

Besides  the  coloring  material  furnished  us  by  fauna  and  flora  there  is  an 
abundance  of  others,  artificially  prepared,  made  synthetically  by  the  chemist. 
They  are  therefore  called  artificial,  although  we  must  always  bear  in  mind  that 
all  dyes,  natural  or  artificial,  can  be  traced  back  to  a  motherbody,  benzol,  so 
that  we  can  take  the  latter  point  of  view  and  deny  any  difference  between  them. 
Accordingly  we  have  already  succeeded  in  preparing  synthetically  some  of  the 
natural  dyes,  e.g.,  indigo. 

Tar  and  Aniline  Dyes. 

All  artificial  dyes  are  made  from  substances  contained  in  tar,  and  are  there- 
fore also  known  as  tar  dyes.  The  term  "aniline  dyes"  is  not  comprising 
enough,  since  aniline  is  by  no  means  the  mother  substance  of  all  artificial  dyes. 

Varieties  of  Artificial  Dyes. 

A  rational  classification  of  artificial  dyes  according  to  their  chemical  com- 
position gives  rise  to  considerable  difficulty.  The  substances  in  which  we  are 
interested  are  best  divided  into  basic,  acid  and  indifferent  dyes. 

Basic  Dyes 
Basic  Dyes. 

As  basic  dyes  are  known  the  salts  of  color  bases,  principally  those  of  muri- 
atic acid,  but  also  the  salts  of  sulphuric,  nitric,  acetic  and  oxalic  acids.  Most 
of  them  are  more  soluble  in  alcohol  than  in  water.  If  an  alkali  is  added  to  a 
watery  solution  of  a  basic  dye,  the  color  base  will  be  thrown  down,  being,  as  a 
rule,  insoluble  in  water.  Tannic  acid  added  to  a  basic  dye  will  form  insoluble 
tannate.  If  a  basic  dye  in  solution  is  treated  with  a  strong  reducing  agent, 
decolorization  takes  place,  a  leuko-compound  being  formed,  which  may  by 
oxidation  be  again  transformed  into  the  original  dye. 

Basic  dyes  possess  an  extraordinary  affinity  for  the  nuclear  chromatin,  for 
which  reason  they  may  be  called  nuclear  dyes.  Other  structures  affected 
by  them  preeminently  are  cartilaginous  substances,  granules  of  mast  cells  and 
mucus. 

Fuchsin. 

This  is  a  nitric  acid  salt  of  rosaniline,  and  in  commerce  is  also  known  as 
rubin,  or  magenta  red,  occurring  in  the  form  of  small  red  crystals  with  a 
metallic  lustre.  It  is  prepared  with  an  aniline  water  solution,  an  excess  of  the 
dye  being  suspended  in  aniline  water  (see  p.  49)  for  several  days  under 


59 

repeated  vigorous  shaking.  This  solution  will  stain  in  from  ten  to  fifteen 
minutes,  acting  more  rapidly  when  warmed.  After  jstaining,  the  excess  is 
extracted  with  95%  alcohol,  to  which  10%  aniline  may  be  added. 

Methyl   Violet. 

The  nitric  acid  salt  of  hexamethyl  pararosaniline  is  a  metallic  shining  crys- 
talline powder,  dissolving  in  water  and  alcohol  with  the  production  of  a  deep 
blue  color.  In  microtechnique  we  generally  use  in  its  stead  the  impure  gentian 
Violet.  This  stain  is  also  used  in  an  aniline-water  solution.  It  stains  in  from 
ten  to  fifteen  minutes  intensively.  For  washing  we  use  first  95%  alcohol,  but 
when  that  seems  insufficient,  Gram's  reduction  method  must  be  resorted 
to.  One  gram  of  iodine  and  2  gms  of  potassium  iodide  are  mixed  with  a  few 
cubic  centimetres  of  water  and  shaken  until  dissolved,  after  which  enough  water 
is  added  to  make  300  cm3.  A  few  drops  of  this  mixture  are  placed  on  the  slide 
after  decanting  previous  washing  liquids,  and  are  allowed  to  act  until  a  brown 
color  results,  a  few  seconds  being  required ;  then  the  slide  is  quickly  transferred 
to  95%  alcohol.  Here  the  slide  will  again  assume  a  blue  color,  discoloration 
setting  in  at  the  same  time.  If  this  does  not  readily  take  place  another  treat- 
ment with  the  iodine  solution  should  be  given.  By  this  method  of  decoloriza- 
tion,  which  is  an  important  one  in  bacteriology,  a  wholly  pure  chromatin  stain- 
ing is  made  possible,  giving  excellent  results,  for  instance  in  the  recognition 
and  study  of  nuclear  changes. 

Methyl   Green, 

a  derivative  of  the  previous  dye,  forms  a  green  micro-crystalline  powder,  is 
easily  soluble  in  water,  not  so  readily  in  alcohol,  giving  a  deep  green  color. 
One  of  our  best  nuclear  stains,  it  is  seldom  used  alone,  but  generally  in  con- 
nection with  acid  dyes. 

Thionine. 

Thionine,  known  also,  as  Lauth's  violet,  is  one  of  the  oldest  coal-tar  dyes 
belonging  to  the  thiazines.  It  occurs  as  a  brilliant  crystalline  powder,  having 
a  metallic  lustre ;  in  water  it  will  slowly  dissolve  with  production  of  a  blue- 
violet  color.  It  is  used  in  an  0.1%  solution  for  nuclear  staining.  After  stain- 
ing (ten  to  fifteen  minutes)  we  wash  in  95%  alcohol.  Its  principal  importance 
lies  in  the  fact  that  this  dye  stains  metachromatically,  e.g.,  principally  mucus, 
cartilaginous  substances  and  the  mast  cell  granules;  they  are  stained  red, 
while  the  nuclei  appear  blue.  If  this  metachromatic  stain  is  to  be  maintained, 
no  after-treatment  with  alcohol  is  permissible  (see  p.  78),  but  simple  water  is 
used  for  washing,  and  the  specimen  mounted  in  levulose. 

Metliylene  Blue. 

This  most  important  of  all  coal-tar  dyes  is  formed  by  the  entrance  of  four 
methyl  groups  into  the  molecule  of  the  preceding  one.  It  occurs  in  various 
forms  on  the  market.  For  our  purpose  the  chemically  pure  crystallized  methyl- 
ene  blue  of  the  Hoechster  Farbwerke  is  the  most  serviceable.  It  consists  of 
small,  glittering,  brilliant  crystallic  needles,  which  are  quite  soluble  in  water 
and  alcohol,  to  a  small  degree  only  is  it  soluble  in  normal  saline,  Ringer's  solu- 
tion, sea-water.  If  an  alkali  be  added  to  a  solution  of  methylene  blue,  the  latter 


60 

is  broken  up  into  methylene  azure  and  methylene  violet.  If  a  watery  solution 
of  methylene  blue  is  treated  with  a  reduction  agent,  it  will  be  decolorized,  the 
methylene  blue  changing  to  leuko-methylene  blue.  Reoxidation  will  take  place, 
when  the  solution  is  shaken,  by  the  mere  action  of  the  oxygen  in  the  air. 
Living  animal  tissues,  blood  and  urine  will  exert  this  same  reducing  action  on 
methylene  blue. 

Methylene  Blue  for 
Nuclear  Staining. 

Methylene  blue  is  an  excellent  nuclear  stain.  For  such  purpose  it  is  used 
in  a  0.5 — 1%  solution;  staining  takes  from  ten  to  fifteen  minutes,  preferably 
in  the  warmth  of  the  paraffin  oven,  after  which  the  specimen  is  treated  in  95% 
alcohol,  to  which  10%  aniline  can  be  added. 

Methylene  Blue  in  the 
Staining  of  Nerve  Tissue. 

The  greatest  value  of  methylene  blue  lies  in  its  property  of  vital  staining 
of  nerves.  Of  the  divers  methods  previously  discussed,  the  following  will  give 
the  best  and  most  constant  results  for  the  central  nervous  system.  As  con- 
cerns the  material,  not  all  animals  are  equally  well  adapted  to  vital  staining 
with  methylene  blue;  rabbits,  guinea-pigs  and  cats  are  most  suitable.  A  1% 
watery  solution  of  the  above-mentioned  chemically  pure  methylene  blue  is 
filtered  just  before  using,  and  heated  to  body  temperature.  The  animal  is 
anaesthetized  with  chloroform,  the  thorax  quickly  opened  in  the  median  line 
with  bone  shears,  the  pericardium  slit  and  the  left  ventricle  opened  by  cutting 
off  the  apex  of  the  heart.  The  protruding  blood  is  carefully  aspirated  in  moist 
cotton  and  the  thoracic  cavity  cleansed  from  blood  coagula.  We  now  search 
for  the  aorta,  which  emanates  from  the  left  ventricle  closely  behind  the  pulmo- 
nary artery  and  by  means  of  curved  forceps  or,  better  still,  an  aneurism  needle, 
we  place  a  ligature  loosely  around  this  blood-vessel.  As  a  cannula  we  use  a 
glass  tube,  slightly  bent  in  the  middle,  about  10  cm  in  length  and  2 — 3  mm  in 
bore.  The  end  to  be  introduced  is  in  the  form  of  a  bulb  which  terminates  in  a 
point,  so  that  the  ligature  may  not  slip.  Such  a  cannula  can  easily  be  made  in 
any  desired  size  (see  p.  76).  The  point  must  be  well  polished  or  molten  in  a 
way  so  as  not  to  injure  the  vessel  wall.  The  other  end  of  the  cannula  is  at- 
tached to  a  rubber  tube,  which  is  closed  by  a  glass  cock  or  a  pinch  cock.  The 
cannula  as  well  as  the  rubber  tube  are  first  filled  with  Ringer's  solution  by 
aspiration,  so  that  no  air-bubbles  may  be  contained  within ;  the  cock  is  then 
closed  and  the  cannula  introduced  with  the  right  hand  into  the  root  of  the 
aorta  by  piercing  through  the  left  ventricle,  while  the  left  hand  fixes  the  heart 
by  means  of  mouse-toothed  forceps.  When  the  bulb  of  the  cannula  is  seen 
lying  in  the  aorta,  an  assistant  will  tighten  the  ligature  placed  previously* 
fixing  the  cannula  in  place.  If  no  assistant  is  at  hand,  the  cannula  is  intro- 
duced into  the  aorta  as  far  as  possible,  then  releasing  his  hold,  the  operator 
tightens  the  ligature  himself.  For  an  injection  apparatus  we  may  use  a  sim- 
ple funnel  suspended  in  a  filter-stative.  The  funnel  is  connected  with  the  can- 
nula by  a  rubber  tube.  Fig.  25  shows  an  apparatus  which  is  more  efficacious. 
It  consists  of  a  graduated  burette,  provided  with  a  stopcock,  which  is  enclosed  in 
a  wide  glass  cylinder,  the  latter  being  closed  at  both  ends  with  rubber  stoppers. 


61 


A  funnel  is  seen  on  top,  through  which  the  outer  cylinder  can  be  filled  with 
warm  water.  Below  an  opening  leads  into  a  drain  pipe,  which  is  provided  with 
a  pinch-cock.  The  upper  cork  has  a  third  opening  for  tHe  exit  of  air,  when  the 
cylinder  is  being  filled.  The  outer  cylinder  is  filled  with  warm  water,  the  burette 
with  Ringer's  solution,  finally  the  lower  end  of  the  burette  is  opened  to  allow  the 
fluid  to  escape  and  therewith  expelling  all  air,  after  which  it  is  attached  to  the 
rubber  tube  leading  to  the  cannula,  and  all  is  ready  for  the  injection.  This 
has  to  be  done  slowly  and  without  undue  pressure. 
First  the  cannula-cock  is  opened,  after  which  the 
one  guarding  the  contents  of  the  burette  is  re- 
leased. All  spurting  vessels  must  be  caught  with 
forceps  and  tied  off.  Owing  to  the  pressure 
caused  by  the  return  flow  through  the  veins  the 
right  heart  will  become  distended  and  must  now 
be  opened,  which  is  best  accomplished  by  cutting 
through  the  right  auricular  appendix.  In  this 
manner  the  entire  vascular  system  is  flushed  with 
Ringer's  fluid,  until  the  latter,  which  is  constantly 
taken  up  with  cotton  sponges  from  the  right 
auricle,  returns  absolutely  clear.  In  the  case  of  a 
cat  this  will  consume  at  least  300  cm3  of  the  fluid. 
The  burette  is  now  filled  with  the  staining  solution 
and  the  injection  proper  begins.  Here,  too,  we 
should  work  with  the  least  pressure  possible  and 
slowly.  The  burette  should  be  opened  only  so  much 
as  to  admit  a  flow  of  1 — 2  cm3  per  minute.  During 
the  entire  duration  of  the  injection  the  staining 
solution  should  be  kept  at  a  temperature  of  about 
42°,  which  can  be  easily  accomplished  by  draining 
off  the  water  in  the  outer  cylinder  and  replacing 
fresh  warm  water.  As  soon  as  the  return  flow 
from  the  right  heart  takes  on  a  deep  blue  color, 
we  tie  or  clamp  the  auricular  appendix  off,  there- 
with closing  the  vascular  system  once  more.  It  is 
hard  to  say  how  much  of  the  stain  should  be  in- 
troduced, at  any  rate  we  cannot  very  well  overdo  it — 120 — 150 — 200  cm3, 
when  injected  within  two  to  three  hours,  should  suffice  for  a  large  rabbit  or  a 
cat.  After  the  injection  is  completed,  the  animal  is  left  undisturbed  for  an- 
other half  hour,  and  now  the  central  organs  are  excised. 

Protection  of  the  Hands. 

Since  soiling  of  the  hands  with  the  dye  is  unavoidable  during  the  injection, 
and  more  so  during  the  ensuing  preparation,  it  is  a  good  plan  to  protect  them 
by  rubber  gloves,  such  as  are  now  in  wide  use  in  pathologic  laboratories. 

Reduction  and 
Resulting  Blue. 

If  the  injection  has  been  properly  executed,  the  organs  will  appear  per- 
fectly colorless  on  opening  the  skull  or  the  vertebral  column  respectively,  but 


FIG.  25. 

Apparatus  for  the  Injection 
of  Methylene  Blue  Solutions. 


62 

as  soon  as  the  air  comes  in  contact  with  them,  they  will  assume  a  blue  color,  so 
that  gray  and  white  matter  can  be  distinctly  differentiated,  the  former  appear- 
ing deep,  the  latter  light  blue.  The  organs  should  be  sectioned  into  not  exces- 
sively small  pieces,  say  3 — 5  mm  in  thickness  and  transferred  immediately  to 
the  fixing  bath. 

Fixing  of  Methylene  Blue 
Preparations. 

We  fix  with  a  10%  solution  of  ammonium  molybdate.  The  salt  is  pulver- 
ized in  a  mortar,  dissolved  in  hot  water,  and  the  clear  solution  cooled  well 
(3 — 5°).  During  the  process  of  fixing,  which  should  be  carried  on  in  the  cool, 
the  specimens  will  assume  a  still  bluer  hue.  The  molybdic  acid  forms  a  salt 
with  the  methylene  blue  base,  which  is  readily  soluble  in  water,  but  poorly  in 
alcohol.  Hence  the  specimens  should  on  the  following  morning  be  thoroughly 
washed  in  running  water  for  several  hours,  while  the  dehydration  must  be 
shortened  and  should  be  instituted  under  as  low  a  temperature  as  possible. 
Four  grades  of  alcohol  suffice:  50%,  75%,  95%  and  100%.  In  each  of  the 
first  three  mentioned  the  sections  remain  from  three  to  four  hours,  being  re- 
peatedly shaken,  after  which  they  are  placed  in  absolute  alcohol  overnight, 
which  is  changed  once  on  the  next  morning.  During  the  day  they  are  trans- 
ferred to  xylol,  in  which  they  may  remain  for  days. 

To  demonstrate  the  peripheral  nerves  it  is  necessary  in  most  cases,  and 
insures  better  success,  to  expose  these  tissues  to  the  air  for  some  time  before 
fixing  them,  as  such  a  precaution  will  bring  out  the  optimum  of  nerve-staining. 
Coarse  razor  sections  are  made  of  the  thicker  structures,  moistened  with 
Ringer's  solution  and  placed  in  a  moist  chamber.  Now  and  then  we  make  a 
control  examination  under  the  microscope.  If  after  two  hours  no  progress  has 
been  made  in  the  nerve-staining,  further  waiting  will  be  useless. 

Organs  which  have  been  removed  from  the  body  or  severed  limbs  can  of 
course  be  injected  in  a  similar  manner. 

Methylene-Azure. 

This  is  the  name  of  a  dye  which  is  produced  by  an  alkali  acting  on  methyl- 
ene blue,  being  readily  soluble  in  water  with  a  blue  color.  It  is  seldom,  per- 
haps never,  used  alone,  but  in  conjunction  with  eosin  (see  p.  67). 

Cresyl   Violet  R.  B., 

an  oxazin  dye,  coming  into  commerce  as  a  bluish  violet  powder,  dissolving 
with  the  same  color  production  not  so  readily  in  water,  but  very  easily  in 
alcohol. 

The  importance  of  the  dye  lies  in  its  metachromasia,  which  in  this  case  is 
accentuated  and  finely  shaded  to  such  a  degree  that  it  is  second  to  no  other  dye. 
A  concentrated  watery  solution  is  made  and  for  staining  is  diluted  about  ten 
times  with  distilled  water.  Staining  will  take  place  in  from  fifteen  to  thirty 
minutes,  overstaining  being  impossible,  even  should  thin  sections  be  left  for 
twenty-four  hours.  Metachromasia  is  evident  most  beautifully  in  the  fresh 
specimen ;  of  fixed  material,  frozen  sections  should  be  used  exclusively.  Forma- 
lin fixation  is  suited  excellently,  but  sublimate  and  nitric  acid  give  also  good 


63 

results.  After  staining,  the  section  is  washed  in  water  and  mounted  in  levulose 
(see  p.  79).  A  blue  color  will  be  imparted  to  the  cell  protoplasm,  the  inter- 
stitial granules  of  the  muscle  fibres,  reddish  violet  to  the -nuclear  chromatin, 
very  light  red  to  the  collagenous  tissue,  deep  red  to  mucus,  cartilaginous  sub- 
stance, mast-cell  granules,  a  yellowish  red  to  the  nerve-sheaths,  a  pure  yellow 
to  the  haemoglobin  of  the  red  blood  cells. 

Safraninr. 

Safraninc  O.  Here  we  have  a  red  powder,  not  very  soluble  in  water,  more 
easily  in  alcohol.  A  concentrated  solution  in  aniline  water  (see  p.  49)  is  pre- 
pared, in  which  the  sections  are  stained  for  at  least  half  an  hour.  To  differen- 
tiate we  transfer  to  95  %  alcohol,  to  which  a  minute  amount  of  hydrochloric 
acid  may  be  added,  a  few  drops  of  a  \%  solution  to  100  cm3  of  alcohol. 

The  metachromatic  properties  of  safranine  are  not  very  pronounced.  The 
best  results  are  obtained  on  mucus,  cartilage  and  sheaths  of  hair  roots. 

Resorcin  Fuchsin. 

This  dyestuff  deserves  mention  here,  its  chemical  composition  being  little 
understood.  Four  grams  of  resorcin  are  added  to  200  cm3  of  a  1%  solution 
of  fuchsin  and  the  mixture  warmed.  When  the  boiling-point  is  reached,  we  add 
25  cm3  of  liquor  ferri  sesquichlorati,  which  is  immediately  followed  by  a  volumi- 
nous precipitate.  The  mixture  is  allowed  to  boil  for  a  few  minutes,  is  then 
cooled  and  filtered.  The  precipitate,  a  bluish  black,  muddy  mass,  together  with 
the  filter  paper  is  brought  to  the  vessel  first  used,  200  cm3  of  95%  alcohol  are 
poured  over  it,  the  whole  is  heated  to  boiling  over  a  water-bath,  subsequently 
cooled  and  filtered;  4  cm3  of  hydrochloric  acid  are  added  and  enough  95% 
alcohol  to  make  200  cm3. 

The  bluish  black  solution  so  obtained  will  stain  within  fifteen  to  thirty 
minutes,  elastic  fibres  will  appear  deep  blue  black,  connective  tissue  a  very 
weak  blue,  the  basic  substance  of  hyaline  cartilage  reddish  violet.  Excess  of 
dye  is  washed  out  thoroughly  with  95%  alcohol. 

Acid  Dyes 
Acid  Dyes. 

The  dyestuffs  considered  under  this  heading  are  mainly  the  alkaline  salts 
of  color  acids,  with  the  possible  exception  of  one  staining  acid,  which  must 
needs  be  mentioned  here,  namely,  picric  acid.  All  are  invested  with  strongly 
pronounced  acid  properties,  which  are  due  to  the  presence  of  certain  atom- 
complexes  (NO2,  SOnH,  COOH,  OH).  Most  of  them  are  readily  soluble  in 
water  and  stain  wool  directly.  Acid  dyes  may  be  decolorized  by  reduction 
agents,  but  a  reoxidation  thereafter  is  jn  most  cases  impossible. 

While  basic  dyes  show  a  pronounced  predilection  for  the  chromatin  of 
nuclei,  the  acid  dyes  possess  an  affinity  for  protoplasm,  for  the  achromatic 
portions  of  the  nuclei  and  to  connective  tissue,  to  varying  degrees ;  e.g.,  picric 
acid  has  a  greater  affinity  for  the  cell  protoplasm,  while  acid  fuchsin  selects  the 
connective  tissue  for  its  action.  For  this  reason  acid  stains  are  seldom  used 
alone,  but  almost  always  in  conjunction  with  basic  dyes,  either  in  mixtures  or 
by  using  the  succedaneous  process.  The  mixtures  will  be  spoken  of  later;  as 


64 

regards  succedaneous  staining,  the  rule  should  be  followed  to  stain  first  with 
the  nuclear  stain  and  secondarily  with  the  acid  dye,  an  excess  of  which  latter 
can  be  extracted  with  alcohol,  70 — 95%.  Often  the  acid  dye  will  act  as  an 
agent  of  differentiation  on  the  basic,  so  that  the  after-treatment  with  alcohol 
will  extract  more  of  the  latter  dye. 

Picric  Acid. 

We  have  already  been  acquainted  with  trinitrophenol  as  a  fixing  agent ;  it 
plays  a  much  more  important  part  as  a  stain.  Picric  acid  is  an  excellent 
plasma  stain ;  its  main  use  is  for  double  staining  after  carmine  and  haema- 
toxylin.  We  must,  however,  always  bear  in  mind,  that  being  a  monobasic  acid, 
it  will  strongly  attract  the  nuclear  stains  of  our  specimen,  similar  to  hydro- 
chloric and  acetic  acid.  It  is  necessary,  therefore,  to  overstain  in  the  nuclear 
dye,  if  a  good  double  staining  is  desired.  It  is  used  in  a  1%  watery  solution, 
staining  cell  bodies,  connective  tissue,  muscle,  red  blood  cells  intensively  yellow 
in  a  few  minutes. 

Acid  Fuchsin 

is  the  alkaline  salt  of  rosaniline-trisulpho-acid,  the  latter  being  prepared  by 
treating  fuchsin  with  fuming  sulphuric  acid.  It  is  a  dark  red  powder,  easily 
soluble  in  water,  not  quite  so  readily  in  alcohol.  The  watery  solution  is  in- 
stantly decolorized  by  an  alkali. 

Acid  fuchsin  has  attained  great  importance  in  histology,  since  it  forms  a 
component  of  many  important  staining  mixtures.  It  is  also  very  serviceable 
for  staining  after  nuclear  staining  has  been  accomplished,  a  0.05 — 0.1%  solu- 
tion in  water  being  very  efficient  for  this  purpose.  A  possible  overstaining  can 
be  corrected  by  washing  in  hydrant  water.  It  plays  an  especially  important 
part  in  the  demonstration  of  Altmann's  bioblasts. 

Bleu  de  Lyon 

(Lyon  blue)  is  made  also  by  sulphurizing  a  basic  dye  and  occurs  as  a  dark 

blue  powder,  readily  soluble  in  water,  more  difficultly  in  alcohol. 

It  is  well  adapted  for  afterstaining  of  carmine  preparations,  for  which  pur- 
pose it  is  used  in  a  0.1%  solution.  Staining  is  continued  until  the  sections 
begin  to  assume  a  blue  color,  when  we  transfer  them  into  70%  alcohol.  In 
well  fixed  specimens  we  can  demonstrate  nerves  and  differentiate  them  from 
the  surrounding  connective  tissue  by  this  method. 

Light  Green. 

Light  green  is  a  dark  green  powder,  readily  soluble  in  both  alcohol  and 
water.  It  is  used  in  the  same  manner  as  the  preceding. 

Orange   G. 

Orange  G.  is  a  representative  of  that  large  group  of  dyestuffs  known  as 
azo  colors,  which  are  of  great  importance  in  commercial  dyeing.  It  forms 
small  yellowish  red  crystal  plates,  which  are  soluble  in  water  and  to  some  extent 
in  alcohol.  Orange  G.  is  one  of  our  best  plasma  stains.  It  is  especially  used 
after  haemalum  staining,  in  a  1%  watery  solution;  it  stains  in  a  few  minutes. 
The  washing  is  done  in  70%  alcohol. 


65 

Bordeaux  R., 

also  an  azo  dye,  is  a  reddish  brown  powder,  readily  soluble  in  water. 

It  is  made  use  of  in  a  0.2%  watery  solution,  which  must  be  applied  before 
the  nuclear  staining  takes  place;  it  stains  in  about  five  minutes,  after  which 
specimens  are  washed  briefly  in  water  and,  as  previously  stated  (see  p.  57), 
are  subjected  to  haematoxylin-iron  alum.  The  more  the  differentiation  ad- 
vances the  more  will  the  red  color  due  to  the  Bordeaux  reappear. 

Eosin. 

Eosin  is  the  sodium  salt  of  a  color  acid,  tetrabromfluorescin.  In  commerce 
it  is  found  as  a  red  powder,  easily  soluble  in  water.  When  a  mineral  acid  is 
added  to  the  watery  solution,  the  insoluble  color  acid  will  be  precipitated. 

Eosin  is  extensively  used  as  a  plasma  stain,  especially  following  haematoxy- 
lin.  The  simplest  manner  of  using  it  is  to  make  a  concentrated  solution  of  the 
preparation  called  "eosin  yellowish,"  and  diluting  the  same  with  ten  to  twenty 
times  its  volume  of  water.  The  thinner  the  solution, 'the  longer  the  staining 
will  last.  After  staining  we  wash  in  70%  alcohol.  Eosin  is  of  great  value  in 
the  staining  of  blood  and  blood  parasites. 

Indifferent  Stains 

Indifferent  Stains. 

Such  is  the  name  applied  to  a  small  group  of  dyes,  the  most  important 
properties  of  which  are  their  relative  solubilities.  They  are  totally  insoluble  in 
water,  dissolve  with  difficulty  in  alcohol,  but  are  very  readily  soluble  in  fats. 
Chemically  these  dyes  are  indifferent,  i.e.,  they  possess  no  salt-forming  groups. 
Under  this  group  belong,  for  instance,  sudan  III  and  scarlet  R.  (Scharlach  R.), 
both  of  which  are  azo  dyes.  They  are  reddish  brown  or  red  powders,  difficultly 
soluble  in  alcohol. 

Sudan  HI  and  Scarlet  R. 
for  Fat  Staining. 

A  concentrated  solution  is  made  in  70%  alcohol,  the  frozen  sections  are 
taken  from  water  and  placed  in  50%  alcohol  for  a  few  minutes,  then  into  the 
staining  fluid  for  fifteen  to  thirty  minutes,  after  which  they  are  washed  in 
water.  Fat  will  appear  intensively  red,  likewise  the  sheaths  of  peripheral  and 
central  nerves.  This  process  may  be  combined  with  a  nuclear  stain,  e.g., 
hasmalum,  as  well  as  with  a  plasma  stain,  e.g.,  picric  acid.  In  such  a  case  we 
first  stain  with  haemalum,  wash  in  water,  transfer  into  50%  alcohol  and  thence 
into  sudan;  the  specimen  is  then  washed  again  in  water,  stained  in  a  watery 
picric  acid  solution  and  finally  rinsed  with  water.  In  the  after-treatment  we 
must  of  course  avoid  strong  alcohol  and  all  solvents  of  fat. 


Staining    Mixtures, 
and  Heterogeneous. 

As  we  have  previously  observed,  multiple   staining   (excepting  metachro- 
matic  staining)  can  be  accomplished  by  either  using  the  various  dyes  in  succes- 


66 

sion  or  by  staining  with  a  mixture  of  the  same.  Three  possibilities  must  be 
considered  in  the  making  of  such  a  mixture:  The  component  dyes  may  be 
basic,  they  may  be  acid,  or  they  may  partly  be  basic  and  partly  acid.  The  first 
two  varieties  are  easily  prepared;  such  homogeneous  mixtures  are  com- 
pounded of  numerous  basic  and  likewise  a  number  of  acid  dyes.  In  the  third 
variety,  the  heterogeneous  mixtures,  we  are  confronted  with  certain  diffi- 
culties. If  we  mix  the  watery  solutions  of  a  basic  and  an  acid  dye,  a  reaction 
will  take  place,  the  color  acid  of  the  acid  and  the  color  base  of  the  basic  dye 
uniting  to  form  a  new  dye,  which  we  name  neutral.  These  neutral  dyes  are 
generally  insoluble  in  water,  therefore  a  precipitate  will  be  thrown  down.  We 
thus  must  expect  a  precipitate  when  mixing  solutions  of  both  acid  and  basic 
dyes.  In  order  to  bring  such  precipitate  into  solution  we  must  employ  other 
solvents,  such  as  ethyl  and  methyl  alcohol,  acetone  or  methylal.  Another  way 
of  procedure  is  to  use  an  excess  of  the  acid  dye  or  to  add  an  extra  acid  dye. 
What  the  nature  of  the  solution  is  in  the  latter  case  cannot  be  stated  with  cer- 
tainty, the  probability,  however,  is  that  aside  from  the  neutral  dye  it  will  also 
contain  both  the  acid  and  the  basic  dyes  in  their  original  form. 

Of  the  homogeneous  mixtures  only  those  of  acid  dyes  are  of  interest  to  us ; 
they  are  extensively  used  for  afterstaining  following  nuclear  dyes,  in  which 
case  they  will  furnish  an  excellent  differentiation  between  connective  tissue  and 
muscle.  Sections  stained  with  a  heterogeneous  mixture  will  show  the  chromatin 
of  the  nuclei,  the  basic  substance  of  cartilage,  mucus  and  the  granules  of  mast 
cells  in  the  basic  colors,  while  all  other  elements  will  take  up  the  acid  dye  or 
dyes,  as  the  case  may  be. 

Picrofuchsin. 

Here  we  deal  with  a  mixture  of  picric  acid  and  acid  fuchsin ;  it  is  prepared 
by  mixing  45  cm3  of  concentrated  watery  picric  acid  solution  with  5  cm3  of  a 
2%  solution  of  acid  fuchsin.  It  is  used  after  nuclear  staining  with  haemalum 
or  iron-haematoxylin ;  it  acts  in  five  to  ten  minutes,  the  excess  dye  being  ex- 
tracted with  70%  alcohol.  Connective  tissue  will  take  on  a  bright  red  stain, 
while  cell  protoplasm  and  muscle  will  appear  yellow. 

Picro-Indig carmine, 

a  solution  of  indigcarmine  in  picric  acid,  is  prepared  by  dissolving  1  gm  of  the 
former  in  300  cm3  of  a  concentrated  watery  solution  of  the  latter.  This  mix- 
ture is  suitable  for  the  after-staining  of  carmalum,  paracarmine  and  safranine 
specimens.  In  five  to  ten  minutes  it  will  impart  a  brilliant  blue  color  to  con- 
nective tissue,  cell  protoplasm,  and  muscle  taking  on  a  grass-green  hue. 
After  staining,  the  specimens  are  washed  in  70%  alcohol. 

Picrocarmine. 

Its  preparation  necessitates  magnesia  water,  which  we  can  make  by  adding 
well  water  to  magnesia  usta,  leaving  this  mixture  stand  for  eight  days,  vigor- 
ously shaking  it  from  time  to  time,  so  that  we  can  always  be  sure  to  have  an 
excess  of  magnesia  present.  Pulverized  carmine  (0.2  gm)  is  boiled  in  100 
cm3  of  magnesia  water  for  half  an  hour  in  a  flask  loosely  plugged  with  cotton. 
After  cooling  we  filter  and  add  a  few  drops  of  formalin.  100  cm3  of  a  0.5% 
watery  solution  of  picric  acid  with  0.15  gm  of  magnesia  carbonate  are  also 


67 

heated  to  boiling,  cooled  and  filtered.  The  two  solutions  combined  will  give  us 
the  stain  ready  for  use.  Five  to  ten  minutes  will  suffice  to  have  nuclei  stained 
red,  all  other  structures  taking  a  yellow  stain. 

Azureosin  Solution. 

This  stain,  also  known  as  "Giemsa  solution,"  contains  3  gms  of  eosin  and 
0.8  gm  of  methylene-azure  dissolved  in  250  gms  of  glycerine  and  250  gms  of 
methyl  alcohol.  One  drop  of  the  solution  in  1  cm3  of  water  will  give  a  ready 
stain ;  its  main  use  is  for  blood  slides. 

Biondi  Solution. 

This  is  the  name  given  to  a  mixture  containing  one  basic  color,  methyl 
green,  and  two  acid  dyes,  acid  fuchsin  and  Orange  G.  Its  efficiency  depends  on 
the  care  taken  in  its  preparation,  which  is  as  follows :  The  colors  to  be  used 
should  be  ordered  from  the  "Aktiengesellschaft  fur  Anilinfabrikation"  at  Ber- 
lin, the  following  names  being  asked  for:  Methyl  green  N.M.P.,  acid  fuchsin 
S.M.P.,  and  Orange  G.M.P.  Of  the  former  we  take  3.4  gms,  of  the  second  4.2 
gms  and  of  the  third  3.0  gms ;  all  three  are  placed  in  a  small  porcelain  mortar 
and  carefully  triturated,  so  that  a  uniform  powder  results,  which  is  dissolved 
in  100  cm3  of  distilled  water.  If  a  permanent  solution  is  desired,  the  container 
must  be  made  of  the  very  best  Jena  .glass.  The  best  plan  is  to  use  an  Erlen- 
mayer  flask,  place  in  it  the  dry  powder  mixture,  add  the  prescribed  amount  of 
water,  cork  well  and,  shaking  it  repeatedly,  let  the  mixture  stand  on  the 
paraffin  oven  for  two  to  three  days.  After  this  period  complete  solution  will 
have  taken  place,  resulting  in  a  dark  brown  fluid  with  a  slight  red  tinge.  This 
stock  solution,  when  properly  taken  care  of,  will  keep  for  a  long  time,  and 
before  using  must  be  acidified  and  diluted.  The  following  procedure,  though 
approximate  only,  is  to  be  recommended  as  practically  very  efficient  for  acid- 
ulation.  Place  1  cm3  of  acetic  acid  in  a  100  cm3  graduated  tube  and  add  dis- 
tilled water  up  to  the  100  cm3  mark,  mix  well  and  empty  the  glass;  the  traces 
of  acid  adhering  to  the  wall  will  suffice  for  the  acidulation.  In  a  second  smaller 
graduate  5  cm3  of  the  stock  solution  are  placed,  and  rinsing  thoroughly  with 
distilled  water  the  contents  are  transferred  to  the  acidulated  tube,  adding 
enough  rinsing  water  to  bring  the  solution  up  to  50,  and  now  our  stain  is  ready 
for  use ;  it  will  keep  well  for  a  few  days.  As  regards  the  dilution,  one  need  not 
literally  cling  to  the  stated  proportion.  As  far  as  the  material  is  concerned, 
we  must  differentiate  between  frozen  and  paraffin  sections.  In  the  former  case 
nearly  all  fixatives  will  yield  good  results,  e.g.,  formalin,  the  various  corrosive 
sublimate  mixtures,  nitric  acid,  also  chrom-osmium-acetic  acid,  if  the  specimens 
have  not  been  subjected  to  its  action  for  too  long  a  period.  Of  the  paraffin 
sections  only  those  fixed  in  sublimate  or  its  mixtures  are  to  be  recommended  as 
suitable.  Celloidin  preparations  are  only  then  admissable,  if  the  celloidin  has 
been  extracted  prior  to  the  staining.  The  duration  of  the  staining  has  little 
influence  on  the  results ;  ten  minutes  will  suffice ;  again  we  may  stain  for  twenty- 
four  hours  with  impunity.  The  staining  is  followed  by  a  short  wash  with  0.5% 
acetic  and  the  specimens  are  transferred  into  70%  alcohol,  where  thick  color 
clouds  will  be  seen  to  emerge.  The  use  of  the  alcohol  must  not  be  protracted 
for  very  long,  but  as  soon  as  the  section  takes  on  a  red  color,  it  should  be  put 


68 

through  absolute  alcohol  and  thence  transferred  to  xylol.  The  results  obtained 
are  most  excellent.  A  bluish  green  is  imparted  to  nuclear  chromatin,  basic 
substance  of  cartilage  and  mucus ;  a  bluish  violet  to  the  mast-cell  granules ;  a 
red  color  appears  in  the  nucleoli,  the  homogeneous  juice  of  the  nuclei,  the 
centrioles,  spheres  and  achromatic  spindles,  the  cell  protoplasm,  collagenous 
and  elastic  tissues,  contractile  substances,  oxyphilic  cell  granules,  basic  sub- 
stance of  bone,  uncalcified  dentine.  The  haemoglobin  of  the  erythrocytes  will 
appear  orange-colored.  The  shading  of  the  red  is  so  fine  as  to  differentiate  all 
various  elements  among  themselves.  In  facility  and  simplicity  of  execution 
and  variety  of  applicability  this  method  is  not  excelled  by  any  other.  One  dis- 
advantage of  Biondi  solution  stands  out  against  its  many  valuable  properties, 
the  fact  that  specimens  prepared  in  such  manner  will  not  keep  indefinitely. 
This  hampering  objection,  however,  may  be  remedied  by  strictly  observing  the 
rules  for  finishing  and  mounting  microscopic  specimens,  as  laid  down  on  pages 
76-79. 


APPENDIX  TO  METHODS  OF  STAINING 

THE    IMPREGNATION    METHODS 

Essentials  of  Impregnation. 

Metal  impregnation  is  the  term  applied  to  a  process  in  which  we  aim  to 
saturate  a  block  or  a  section  of  our  specimen  with  a  solution  of  a  metal  salt, 
the  latter  undergoing  such  reduction  in  certain  parts  of  the  tissue,  which 
enables  it  to  form  colored  compounds  or  to  be  changed  to  the  metal  itself,  re- 
spectively. Thus  the  metal  might  be  precipitated  in  these  places  as  fine 
granules,  or  these  certain  tissue  elements  may  appear  distinctly  colored,  with- 
out any  precipitate  being  formed.  In  the  latter  case  a  differentiation  between 
impregnation  and  staining  is  impossible. 

Metals  Concerned. 

Of  the  metals  used  for  this  purpose  only  the  noble  metals  and  of  these  only 
silver  and  gold  will  interest  us.  Another  impregnation,  that  with  osmic  acid, 
has  been  discussed  among  the  fixation  methods  and  impregnation  in  that  case, 
when  applied  to  fresh  tissues,  naturally  serves  as  a  fixation  as  well,  since  the 
salts  of  the  noble  metals  will  all  coagulate  albumin. 

The  metal  salt  solution  may  be  allowed  to  act  on  either  the  fresh  speci- 
men or  the  fixed  specimen  or  finally  on  sections  made  from  the  latter.  Each  of 
these  three  methods  will  answer  the  purpose. 

The  Reduction 
of  the  Metal  Salt. 

Reduction,  the  most  important  phase  in  the  process  of  impregnation,  may 
be  induced  without  our  aid  by  the  tissue  itself;  ordinarily,  however,  we  aug- 
ment the  process  by  the  use  of  some  aiding  factors.  One  such  factor  is  light — 
diffuse  daylight  or  the  direct  rays  of  the  sun.  Under  its  influence  the  changes 
will  take  place  much  more  rapidly  and  more  intensely.  Another  such  factor  is 
found  in  acidulation ;  the  reduction  does  not  take  place  in  distilled  water,  but 


69 

to  the  latter  is  added  a  greater  or  lesser  amount  of  acid.  Warming  may  in 
certain  instances  also  hasten  reduction.  Finally  we  resort  in  many  cases  to 
measures  such  as  used  by  the  photographer  in  the  developing  «f  plates,  namely, 
to  the  use  of  strong  reducing  agents,  e.g.,  sulphurous  acid,  arsenious  acid,  for- 
maldehyde, resorcin,  aniline,  pyrogallic  acid,  hydrochinon,  etc. 

Frequently  we  imitate  the  photographer  so  far  as  to  follow  our  reduction 
with  a  hyposulphite  of  soda  bath,  to  remove  any  metal  salt  which  has  not  been 
reduced. 

Impregnation  with 

Silver  Salts.    Silver  Nitrate. 

Of  the  silver  salts  the  only  one  of  importance  is  silver  nitrate.  In  com- 
merce we  find  it  either  in  the  form  of  crystals  or  in  sticks ;  it  may  be  dissolved 
in  water  to  the  extent  of  200%,  forming  a  strongly  corrosive  fluid  with  neutral 
reaction.  A  stock  solution  of  20%  strength  should  be  made  and  kept  in  a 
clean,  glass-stoppered  bottle.  If  impurities  are  present,  silver  salt  will  soon 
be  thrown  down. 

Silvering. 

A  solution  of  0.5 — 1%  (0.5 — 1  cm3  of  our  stock  solution  to  20  cm3  of  water) 
will  suffice  to  silver  thin,  membranous  sections.  The  membranous  tissue  is  taken 
from  the  animal,  spread  quickly  on  a  flat  plate,  which  is  lined  with  paraffin  and 
then  fastened  with  hedgehog  bristles;  it  is  carefully  rinsed  just  once  with  dis- 
tilled water.  According  to  the  size  of  the  specimen  10 — 20  cm3  of  the  silver 
solution  are  poured  over  the  sections  and  spread  evenly  by  slight  and  even  bal- 
ancing over  all  the  parts,  which  latter  gradually  become  opaque.  After  ten 
minutes  the  solution  is  decanted,  and  we  rinse  in  water  several  times,  fill  the 
plate  with  acidulated  (1%  acetic  acid)  water  and  expose  it  to  light,  preferably 
sun-rays,  for  reduction.  The  latter  will  take  place  very  rapidly,  the  specimen 
becoming  gradually  brown.  This  silvering  process  preeminently  brings  out 
the  cement-substance,  being  the  classic  method  for  that  purpose. 

Another  use  to  which  silver  nitrate  is  put  is  the  injection  of  the  solution 
into  hollow  structures,  such  as  the  vascular  system,  lungs,  etc.  The  technique 
will  be  described  in  the  special  part. 

Chrome  Silver  Method. 

The  Golgi  method,  so  called  after  its  discoverer,  consists  in  the  successive 
treatment  of  the  specimen  with  a  chrome  salt  and  silver  nitrate  solution.  It  is 
fair  to  assume  that  through  the  action  of  the  bichromates  on  the  tissues,  chrom- 
albumins  are  formed,  the  composition  of  which  depends  a  great  deal  on  the 
duration  of  the  process.  If  the  specimen  is  now  brought  into  a  silver  nitrate 
solution,  a  compound  of  silver  albumin-bichromate  is  formed  in  certain  tissue 
parts,  appearing  black  when  viewed  before  a  light.  We  will  thus  find  these 
parts  prominently  black  in  a  yellow  field.  Not  all  the  tissue  elements  of  the  same 
kind  are  equally  impregnated,  since  this  'process  favors  a  certain  amount  of 
them,  while  it  utterly  disregards  others.  Besides  this  regular  impregnation  the 
entire  surface  of  the  specimen  and  some  of  the  deeper  parts  wijl  be  found 
studded  with  granular  or  crystalline  deposits,  which  act  very  disturbingly. 


70 

Rapid  Chrome-Silver 
Method. 

Of  the  many  variations  of  the  method  the  rapid  method  gives  the  best 
results,  and  we  will  therefore  devote  special  attention  to  it.  The  fresh  speci- 
mens are  cut  into  small  pieces  and  placed  in  a  mixture  of  8  parts  of  2.5%  bi- 
chromate of  potassium  and  2  parts  of  1%  osmic  acid  for  two  to  five  days ;  they 
are  then  dried  with  tissue  paper  and  transferred  to  a  0.75%  solution  of  silver 
nitrate,  which  must  be  changed  after  a  few  minutes.  They  will  be  ready  for 
cutting  the  next  day.  The  following  details  may  well  be  observed : 

Material  for  the 
Chrome-Silver  Method. 

Mostly  every  organ  is  suited  for  the  chrome  silver  method,  and  in  every  one 
some  part  or  other  will  be  stained  black :  Connective  tissue  shreds,  elastic 
fibres,  muscle  fibres,  capillaries,  neuroglia  cells,  nerve-endings,  etc.,  but  fore- 
most among  all  others  nerve-cells  and  their  branches,  peripheral  and  central 
nerve-fibres.  The  importance  of  this  method  rests  with  this  property,  and  for 
this  reason  its  main  use  is  found  in  the  study  of  the  central  and  peripheral 
nervous  system.  The  material  should  be  as  fresh  as  possible ;  however,  we  fre- 
quently find  this  requisite  greatly  exaggerated.  Six  to  twelve  hours  after 
death  we  will  still  have  tissues  which  give  excellent  results. 

A  more  important  fact  is,  especially  when  dealing  with  the  central  organs, 
that  only  very  young  specimens  can  be  used.  When  human  material  is  desired, 
infants  one  week  old  are  to  be  recommended ;  of  animals :  mice,  dogs,  cats,  two 
to  ten  days  old,  guinea-pigs  at  full  term.  The  central  organs  of  the  calf  are 
excellent,  if  they  can  be  procured  early  enough. 

Moderately  sized  pieces,  not  too  small,  are  selected,  perhaps  2 — 3  mm  in 
thickness  and  5 — 10  mm  long.  Several  pieces  should  be  taken  from  each  speci- 
men and  all  be  placed  in  a  brown  vessel  containing  50  cm3  of  fixing  solution. 

Duration   of   Impregnation. 

It  is  of  vital  importance  to  know  how  long  the  pieces  should  remain  in  the 
solution.  There  are  no  set  rules  for  each  organ,  the  process  being  empirical. 
On  the  morning  of  the  third  day  the  pieces  are  dried  superficially  with  filter- 
paper  and  thereafter  placed  in  10  cm3  of  silver  solution,  where  a  red  precipi- 
tate of  silver  bichromate  will  instantly  be  formed.  After  a  few  minutes  fresh 
solution  is  added  liberally  until  the  fluid  remains  clear.  The  following  morn- 
ing some  razor  sections  are  made  and  examined.  If  the  impregnation  is  well 
established,  the  entire  material  can  be  silvered  in  like  manner.  If  the  result 
was  not  satisfactory,  a  few  more  pieces  are  selected  and  experimented  on,  etc. 
After  seven  or  eight  days  a  success  is  improbable. 

Further  Treatment  of 
Impregnated  Specimens. 

Embedding  can  usually  be  omitted.  The  following  is  the  best  after-treat- 
ment. After  remaining  in  the  silver  solution  for  from  twenty-four  to  twenty- 
eight  hours,  the  pieces  are  briefly  rinsed  in  distilled  water  and  in  groups  are 
placed  at  a  proper  height  upon  stabilit-blocks  (p.  47),  with  a  thick  solution 
of  gum  arabic.  After  a  few  minutes  the  blocks  are  immersed  in  70%  alcohol, 


71 

after  a  few  hours  in  a  95%  solution  of  alcohol,  where  within  a  few  hours  they 
will  attain  the  proper  cutting  consistency.  The  next  day  sectioning  with  the 
microtome  may  begin.  The  knife  is  placed  at  an  obtuse  angle  and  diligently 
moistened  with  95%  alcohol;  the  sections,  which  should  not  be  too  thin,  are 
transferred  to  95%  alcohol.  They  are  then  transferred  to  carbolxylol  (see  p. 
79),  and  finally  to  pure  xylol,  where  they  may  remain  a  long  time  with  im- 
punity. 

Mounting. 

The  mounting  cannot  take  place  in  the  usual  manner,  as  will  be  described 
later;  they  will  soon  spoil  if  so  treated.  They  are  subjected  to  the  following 
technique.  Cover-glasses  of  corresponding  numbers  and  sizes  are  cleansed  and 
placed  on  a  specimen  board;  the  sections  are  taken  from  the  xylol  and  placed 
on  the  cover-glasses,  leaving  a  good  margin  of  the  glass  free.  As  soon  as  the 
xylol  begins  to  evaporate,  a  large  drop  of  Canada  balsam  is  placed  on  each 
section  and  the  latter  put  in  a  dust-free  place  to  dry.  The  following  day  the 
specimens  are  looked  over  and  if  any  have  risen  in  the  balsam,  another  drop  is 
applied.  After  a  few  days  the  specimens, 
now  completely  dry,  are  placed  upon 
slides,  which  may  be  made  of  cardboard, 
wood,  metal  or  glass,  an  opening  being  cut 
out  in  the  place  where  the  specimen  is  to 
rest  (Fig.  26).  Such  slides  can  be  bought, 

but  on  the  other  hand  are  very  easily  made         Wooden  Slide  for  Chrome-Silver 
from   cigar-box  wood  with   a  little   inge-  Specimens, 

nuity.     The  cover-glass  is  placed  in  the 

opening,  specimen  side  down,  and  secured  with  a  little  cement  (see  p.  77).  If 
mounted  in  this  fashion,  specimens  will  keep  for  a  long  time  and  can  even  be 
examined  with  the  oil-immersion  lens. 

The  Pyrogallol  Method. 

We  will  give  this  name  to  the  method,  since,  similar  to  the  photographer, 
we  use  pyrogallic  acid  to  create  the  silver  image  in  the  tissues.  Specimens  are 
placed  in  90%  alcohol  overnight,  8  drops  of  spirits  of  ammonia  (specific 
gravity,  0.91)  having  been  added  to  each  100  cm3  of  alcohol;  rinse  with  water 
and  transfer  to  a  2%  solution  of  silver  nitrate  for  five  to  eight  days,  preferably 
in  a  thermostat  to  keep  the  temperature  even.  The  pieces  are  now  washed  in 
water  again  and  brought  into  the  following  reduction  mixture :  pyrogallic  acid, 
1  gm;  formalin,  10  cm3;  water,  100  cm3.  After  twenty- four  hours  they  are 
dehydrated  in  the  usual  manner  and  embedded  in  paraffin.  This  method  gives 
good  results  for  the  study  of  the  neuro-fibrils  in  nerve-cells  and  fibres.  It  is 
best  suited  for  the  central  nervous  system,  less  efficient  for  the  peripheral.  The 
paraffin  sections,  mounted  in  the  usual  manner,  can  be  gilded  by  placing  them 
in  a  0.1%  gold  chloride  solution  for  several  minutes,  succeeded  by  ten  minutes' 
treatment  in  5%  fixing  soda,  after  which  they  are  washed  in  water. 

The   Silver- Ammonia   Method 

can  be  used  for  frozen  sections  as  well  as  for  entire  tissue  blocks  previously 

fixed  in  formalin.     We  will  only  discuss  the  latter.     The  blocks  are  washed  in 


distilled  water  for  twenty-four  hours,  impregnated  in  a  2%  silver-nitrate  solu- 
tion for  six  to  eight  days,  quickly  rinsed  in  water  and  placed  in  the  following 
solution  of  silver-ammonia,  which  must  be  freshly  prepared ;  20  cm3  of  a 
2%  silver  nitrate  solution  are  placed  in  a  large  beaker  with  3  drops  of  4>0% 
sodium  hydroxide,  shaken  and  the  resulting  precipitate  of  silver  oxide  is  dis- 
solved by  adding  ammonia  drop  by  drop  until  the  solution  is  absolutely  clear. 
The  brown  staining  of  tissues  will  be  materially  intensified  by  this  solution. 
The  following  morning  we  wash  once  more  and  transfer  to  20%  formalin  for 
reduction. 

This  method  gives  similar  results  as  the  one  preceding.  The  collective 
staining  of  the  connective  tissue  is  a  disadvantage. 

Gold   Chloride. 

Impregnation  with  Salts  of  Gold. 

Gold  chloride  is  the  salt  used  almost  exclusively  in  microtechnique.  In 
commerce  we  find  it  in  the  form  of  reddish-brown  amorphous  lumps  or  as  yellow 
crystalline  needles.  The  chemical  composition  of  the  former  is  AuCl3+2H2O, 
while  the  latter  forms  a  compound  with  hydrochloric  acid  and  is  represented 
by  AuCl3-|-HCl-j-3H2O.  Both  salts  are  highly  hygroscopic,  hence  they  are 
sold  in  sealed  glass  tubes.  Of  either  one  of  the  salts  a  2%  stock  solution  is 
prepared  and  kept  in  a  well-stoppered  white  bottle.  If  cleanliness  is  observed, 
it  will  keep  indefinitely.  In  the  handling  of  all  gold  solutions  metal  instruments 
must  be  avoided,  wooden  sticks  or  glass  needles  being  used  in  their  stead ;  the 
latter  can  easily  be  made  over  the  gas-flame  from  barometer  tubes  and  drawn 
out  to  any  desired  shape  or  thickness. 

Of  all  the  numerous  gilding  methods,  which  have  been  advocated  in  the 
course  of  time,  we  will  only  discuss  three. 

Gold  Chloride- 
Lemon-Juice  Method. 

This  method  is  especially  adapted  for  thin  organs,  rich  in  connective  tissue. 
The  pieces,  according  to  their  thickness,  are  placed  for  from  five  to  fifteen 
minutes  in  freshly  expressed  lemon- juice,  which  contains,  besides  its-  citrates,  a 
goodly  amount  of  free  citric  acid.  The  specimens  will  be  found  to  swell  con- 
siderably; they  are  now  rapidly  washed  in  water  and  placed  in  1%  gold 
chloride  for  a  half  to  one  hour.  Again  they  are  rinsed  and  then  reduction  is 
induced  by  placing  them  into  \%  acetic  acid,  preferably  on  a  white  background 
and  exposed  to  the  direct  rays  of  the  sun.  When  taken  from  the  gold  solution 
the  pieces  present  a  light  or  dark  yellow  color;  exposed  to  the  light  they  will, 
after  a  shorter  or  longer  period,  assume  first  a  dirty  brown,  later  a  red,  and 
finally  a  bluish  violet  hue,  depending  on  the  intensity  of  the  light.  The  last 
color  mentioned  might  become  so  deep  that  the  specimen  appears  black. 
Axiscylinders  are  most  prone  to  be  affected  by  the  reduction,  appearing,  in  a 
good  specimen,  distinctly  black  on  a  brilliant  red  background. 

In  order  to  make  sections,  the  pieces  are  subjected  to  5%  formalin, 
and  later  are  cut  with  the  freezing  microtome.  Such  preparations  will  last 
well. 


Gold   Chloride- 
Formic  Acid  Method. 

A  process  most  suited  to  the  demonstration  of  muscle  nerves.  Thin  layers 
of  the  muscle  are  spread  and  fastened  on  wax  plates  and  placed  in  30%  formic 
acid  for  ten  to  twenty  minutes.  We  follow  with  1%  gold  chloride  for  one-half 
to  one  hour,  and  finally  reduce  with  the  previously  named  acid  in  the  dark. 
Reduction  will  be  largely  completed  the  next  day.  We  now  transfer  to  glycer- 
ine which  must  contain  the  same  acid  to  the  extent  of  50%  by  volume,  and 
substitute  pure  glycerine  on  the  following  day,  which  will  act  as  a  Dreservative. 

Gold  Chloride-Iodine- 
lodine-Potassium  Method. 

In  conclusion  we  will  deal  with  this  process,  which  is  used,  not  on  fresh,  but 
on  preserved  material,  viz.,  sections.  The  gilding  of  such  sections,  whether 
frozen  or  paraffin,  will  always  offer  difficulties  inasmuch  as  they  must  primarily 
be  rendered  susceptible  to  the  gold  salt.  Frozen  sections,  fixed  in  any  of  the 
methods,  are  first  placed  in  an  iodine-iodine-potassium  solution,  made  by 
diluting  our  laboratory  solution  (see  p.  59)  with  an  equal  part  of  water,  where 
they  remain  for  thirty  minutes.  They  will  take  on  a  prominent  reddish  brown. 
We  now  rinse  them  briefly  in  water  and  transfer  them  to  a  0.2%  solution  of 
gold  chloride  for  thirty  minutes,  where  they  are  at  first  decolorized  and  later 
assume  a  yellow  color.  After  washing  again  redaction  takes  place  in  a  2% 
solution  of  resorcine.  Reduction  will  be  complete  in  from  one  to  two  hours, 
but  may  be  interrupted,  if  desired.  Rinse  in  water  and  place  in  a  5%  solution 
of  sodium  hyposulphite,  which  will,  in  the  course  of  fifteen  to  thirty  minutes, 
dissolve  out  all  the  unreduced  gold.  The  sections  are  finally  thoroughly 
washed.  This  method  gives  excellent  results  for  many  purposes,  but  chiefly 
serves  to  demonstrate  connective  tissue  fibres,  neuroglia  giving  the  next  best 
results. 


METHODS   OF   INJECTION 

As  an  appendix  to  general  microtechnique  the  technique  of  injection  de- 
serves special  reference  at  this  juncture,  a  process  which  the  microscopist  has 
largely  borrowed  from  the  art  of  macroscopic  procedure.  By  "injection"  we 
understand  the  filling  of  any  hollow  structures  of  the  animal  body  with  foreign 
material  by  the  aid  of  proper  instruments. 

The  spaces  may  be  of  the  most  varied  kind,  e.g.,  the  communicating 
channels  of  bony  structure,  joint  cavities,  and  the  excretory  ducts  of  glands. 
As  a  rule,  however,  the  term  injection  is  applied  exclusively  to  the  filling  of  the 
blood  and  lymphatic  systems. 

Material    Used  for  Injection. 

As  injection-material  we  can  use  all  sorts  of  matter,  which  must  needs 
be  liquid  (or  gaseous)  at  the  time  of  injection.  For  obvious  reasons  we  select 
generally  a  colored  injection  material.  They  can  be  classified  in  different  ways 
and  may  be  spoken  of  as  unstained  and  stained  ,*  the  latter  can  be  again 
divided  into  opaque  and  transparent.  We  can  furthermore  differentiate  be- 


74 

tween  liquid  (i.e.,  remaining  liquid)  and  coagulating.  Liquid  material  is 
chosen  for  the  injection  of  very  fine  blind  ducts,  such  as  the  excretory  ducts  of 
glands,  while  coagulating  fluids  are  better  suited  for  blood-vessels. 

Properties  of  an  Efficient 
Injection  Material. 

When  a  colored  mass  is  to  be  used,  it  must  contain  the  particular  color  in 
an  indiffusible  form,  so  that  it  will  not  diffuse  through  the  vessel-walls,  staining 
the  proximal  tissues  and  thereby  rendering  the  preparation  useless.  Hence 
dyes,  which  are  insoluble  in  water,  can  always  be  used  to  advantage,  provided 
we  use  a  very  fine  suspension  thereof.  Soluble  dyes  give  much  nicer  results,  but 
of  these  only  very  few  are  not  diffusible ;  here  we  can  mention  the  soluble  Berlin 
blue  and  the  carmine,  the  latter,  however,  only  under  certain  conditions. 

Soluble  Berlin  Blue. 

A  concentrated  watery  solution  of  the  soluble  Berlin  blue,  previously 
filtered,  makes  the  best  watery  injection  material  for  glandular  ducts. 

For  the  injection  of  blood-vessels  we  always  use  a  coagulating  mass,  i.e.,  a 
mass  which  is  injected  warm  and  liquid,  coagulating  when  it  becomes  cooled. 
For  this  purpose  a  sufficient  amount  of  glue  is  added  to  the  color  solution. 
Such  a  glue-mass  coagulates  easily  and  completely,  and  will  not  interfere  with 
subsequent  microtechnical  procedures. 

The  Blue   Glue-Mass. 

An  efficient  blue  glue-mass  is  prepared  as  follows :  50  gms  of  the  best 
gelatine  are  soaked  in  distilled  water  for  several  hours ;  the  water  is  thoroughly 
decanted  at  the  expiration  of  this  time  and  the  gelatine  placed  in  a  beaker  of 
1,000  cm3  capacity,  and  warmed  over  a  water-bath  until  completely  liquid. 
500  cm3  of  a  concentrated  solution  of  Berlin  blue  of  the  same  temperature  are 
carefully  poured  into  the  gelatine,  constantly  stirring  and  raising  the  tempera- 
ture until  all  of  the  dye  has  been  dissolved.  The  hot  mass  is  then  filtered 
through  flannel  with  the  aid  of  a  hot-water  funnel. 

The  Red  Glue-Mass. 

A  good  red  glue-mass  is  made  by  using  50  gms  of  gelatine,  leaving  it  to 
swell  in  distilled  water  for  several  hours,  after  which  time  we  decant  the  water 
carefully.  The  gelatine  is  now  stained  for  two  to  three  days  in  a  solution 
of  15  gms  of  carmine  in  2  I  of  10%  borax  solution.  The  gelatine  bars,  after 
being  soaked,  should  be  transferred  singly  to  the  solution.  After  the  staining 
the  excess  dye  solution  is  washed  off  with  hydrant  water,  and  again  the  plates, 
now  deep  blue  red,  are  singly  transferred  to  a  vessel  containing  2%  hydro- 
chloric acid.  The  bars  are  agitated  in  this  solution,  until  the  bluish  red 
changes  to  a  cherry  red.  Subsequently  they  are  freed  from  the  acid  by  washing 
in  running  water ;  all  water  is  removed  or  evaporated  over  the  water  bath.  For 
purposes  of  preservation  a  piece  of  camphor,  the  size  of  a  hazel  nut,  is  added 
to  the  mass,  while  hot. 

The  Injection  Syringe. 

As  an  instrument  for  injection  we  recommend  for  most  cases  a  strong  metal 
syringe,  holding  100 — 200  cm3,  the  piston  of  which  should  have  a  washer  of 


75 


leather  or  asbestos  (Fig.  27).  The  anterior  conical  extremity  serves  for  the 
attachment  of  an  intermediate  piece  with  stopcock,  preferably  a  glass  cannula, 
instead  of  the  impractical  metal  cannula,  as  depicted 
in  our  illustration. 

Preparation  of  Glass  Cannulce. 

These  can  be  made  in  any  desired  form  or  size,  to 
suit  each  particular  case,  in  the  following  manner 
(Fig.  28).  A  glass  tube  of  the  proper  calibre  is 
selected  and  heated  on  one  spot  over  a  small  gas- 
burner  (microburner  so-called),  the  tube  being  con- 
stantly rotated.  When  the  glass  has  softened,  it  is 
drawn  out  according  to  how  thin  a  cannula  is  de- 
sired. The  flame  is  now  turned  very  small  and  ro- 
tating, we  again  heat  on  the  spot  designated  in  Fig. 
28  a,  draw  out  slightly,  thus  molding  the  neck  of  the 
cannula ;  now  we  heat  on  the  place  shown  in  Fig.  28  b 
and  draw  out  completely,  so  that  the  head  terminates 
in  a  point.  The  point  is  now  severed  closely  behind 
the  head  with  the  glass  file  and  the  opening  polished 
smooth  on  a  hone.  The  cannula  shown  in  Fig.  28  c 
would  answer  better  for  many  purposes  if  it  would 
come  to  a  finer  point. 

The  cannula  is  connected  with  the  intermediate 
piece  by  means  of  a  strong  rubber  tube,  which  is 
secured  on  both  ends  with  strong  silk  or  thin  wire. 

To  inject  cold  liquids  the  apparatus  described  on 
p.  61  can  be  utilized.  By  raising  and  lowering  the  en- 
tire apparatus,  the  pressure  can  be  regulated  at  will. 

Technique  of  Injecting. 

It  can  be  stated,  as  a  rule  governing  all  injec- 
tions, that  the  smaller  the  area  to  be  injected,  the 
better  will  be  the  results  obtained.  Total  injection 
of  an  entire  animal  body  requires  much  care  and  ex- 
perience. If  a  glue  mass  is  to  be  injected,  the  speci- 
men must  have  a  temperature  above  the  melting-point 

of  the  mass.  Cold  parts  of  a  body  must  therefore  be  warmed  for  hours  in 
water  of  40 — 45°.  When  using  recently  killed  animals,  this  is,  of  course,  un- 
necessary. If  the  object  is  warm,  we  first  introduce  the  cannula. 

The  Introduction 
of  the  Cannula. 

A  double,  strong  silken  thread  is  placed  around  the  vessel  and  left  untied. 
After  severing  the  latter,  one-half  is  left  in  the  hand  of  the  operator  to  serve 
as  a  guide,  while  the  assistant  makes  a  loose  loop  of  the  other  half.  The 
operator  now  opens  the  vessel  along  its  longitudinal  axis  to  the  extent  of  about 
5  mm  and  introduces  the  cannula,  which  has  previously  been  connected  with 
rubber-tube  and  intermediate  piece  and  filled  with  warm  salt  solution  or,  better 


FIG.  27. 
Injection  Syringe. 


76 

still,  with  an  8%  solution  of  Glauber  salt,  all  air-bubbles  being  expelled  and  the 
stopcock  being  closed  at  the  time  of  introduction ;  as  soon  as  the  head  of  the 
cannula  is  within  the  vessel,  the  assistant  secures  it  by  tying  the  previously 
placed  ligature.  We  can  now  inject  our  solution,  which  must  be  warmed  to 
40°,  or  we  may  first  remove  blood  and  clots  by  injecting  a  warm  8%  solution 
of  Glauber  salt  and  then  follow  with  the  injection  mass.  By  the  latter  pro- 
cedure we  warm  our  specimen  and  obtain  a  more  complete  injection;  of  course 
more  of  the  mass  is  needed. 

As  soon  as  the  injection  material  is  seen  to  come  from  the  efferent  vessels, 


FIG.  28. 
Preparation  of  a  Glass  Cannula. 

the  latter  are  closed  with  artery  clamps  (Fig.  29),  and  we  continue  carefully 
with  our  injection  until  the  entire  vascular  system  appears  distended.     It  is 


FIG.  29. 
Artery  Clamps. 

a  matter  of  practice  and  experience  to  know  just  when  to  discontinue  the 
injection  and  how  much  pressure  to  use  during  it. 

After-Treatment. 

This  is  much  the  same  as  the  routine  with  which  we  are  familiar.  The 
injection  being  complete,  we  cool  the  specimen  in  order  to  coagulate  the  solu- 
tion, which  may  require  hours  in  the  case  of  voluminous  organs.  Smaller  pieces 
are  subsequently  excised  and  fixed  with  any  method  desired,  formalin  par 
excellence. 

MOUNTING  AND  FINISHING  OF  THE  MICROSCOPICAL 

SPECIMEN 

Durability  of 
Stained  Specimens. 

After  having  stained  our  specimen  according  to  one  of  the  methods  de- 
scribed, the  question  arises  how  to  preserve  it  in  a  manner  which  is  least  apt  to 
allow  any  changes  to  take  place  and  will  make  the  specimen  a  durable  one.  In 


77 

many  cases  this  remains  a  hope,  since  among  all  our  dyes  there  are  few  only 
of  which  it  can  be  said  that  specimens  stained  therewith  are  absolutely  lasting 
in  color.  Perhaps  we  can  rightfully  claim  this  for  carmine  and  haematoxylin, 
but  most  all  will  be  bleached  in  the  course  of  years,  and  this  is  especially  true 
of  the  artificial  dyestuffs. 

A  great  deal  depends  on  the  mounting  and  care  of  the  slide  thereafter.  Any 
agent,  to  serve  for  mounting,  must  be  chemically  pure  and  above  all  must  not 
contain  a  trace  of  any  acid,  which  would  destroy  any  stain  in  the  course  of 
time.  Many  artificial  dyes  being  only  slightly  fast  to  light,  specimens  should 
be  protected  from  light. 

Mounting  Media. 

The  agents  used  to  enclose,  i.e.,  mount  our  specimens,  mounting  media  so- 
called,  are  partly  liquid,  partly  solid.  In  the  latter  case  they  must  naturally 
be  liquefied  by  a  proper  solvent  before  using ;  by  spontaneous  or  induced  evapo- 
ration of  this  solvent  the  specimen,  covered  by  the  cover-glass,  is  thus  enveloped 
in  a  layer  of  the  solid  medium,  which  serves  also  as  a  cement  between  slide  and 
cover-glass. 

Mounting  in  Liquid  Media. 

When  using  a  liquid  medium,  the  latter  must  be  protected  against  evapo- 
ration, i.e.,  the  specimen  must  be  prevented  from  drying  up,  by  the  use  of  a 
strong  cement  between  slide  and  cover-glass. 

Cover-Glass  Cement. 

Such  a  cement  can  be  prepared  by  melting  10  gms  of  wax  in  a  porcelain 
dish  and,  while  stirring,  adding  35  gms  of  rosin.  To  make  the  mass  more 
pliable  a  small  amount  of  turpentine  may  be  added.  The  mixture  should  be 
applied  with  a  broad  and  not  too  thin  spatula.  The  spatula  should  be  warmed 
first  and  then  dipped  in  the  solid  mass;  the  necessary  amount  of  cement  will 
thus  adhere  to  the  spatula  and  the  latter  can  be  heated  to  such  a  degree  as  to 
render  the  cement  thinly  liquid.  The  groove  formed  by  cover-glass  and  slide  is 
touched  with  the  edge  of  the  spatula,  and  thus  a  band  of  cement  can  be  drawn, 
which  solidifies  at  once.  The  surface  to  which  the  cement  is  applied  must  be 
absolutely  clean  and  dry,  lest  the  cement  will  not  adhere.  It  is  important  there- 
fore to  use  a  small  drop  of  mounting  fluid,  so  that  none  will  be  protruding  from 
under  the  edges  of  the  cover-glass. 

Mounting  in  Solid  Media. 

The  process  is  simpler,  when  using  solid  media.  A  small  drop  of  the 
liquefied  medium  should  be  used,  for  the  smaller  the  drop,  the  thinner  will  be  the 
layer  enveloping  the  section.  The  cover-glass  is  grasped  with  forceps,  the  free 
edge  is  approximated  until  it  touches  the  slide  and  the  glass  then  slowly 
lowered,  being  careful  not  to  enclose  any  air-bubbles.  The  specimen  is  then 
left  to  dry  in  a  dustproof  place.  In  certain  instances  it  is  of  advantage  to 
hasten  the  drying  by  the  use  of  the  thermostat. 

Thickness,  Size  and 
Cleansing    of   the    Cover-Glass. 

The  cover-glasses  with  which  we  cover  our  specimens  must  be  sufficiently 
large  to  cover  the  tissue  entirely.  The  latter  must  never  come  close  to 


78 

the  edge  of  the  glass,  lest  it  shall  be  bleached  more  readily.  This  is  especial- 
ly true  of  haematoxylin  preparations.  The  size  of  the  cover-glass  is  naturally 
dependent  upon  the  size  of  the  slide,  the  latter  generally  measuring  26  X  "76 
mm,  the  so-called  English  type.  In  order  to  leave  a  narrow  margin,  the  cover- 
glass  must  therefore  not  exceed  20  X  22  mm.  Generally  those  measuring  20 
mm  square  are  well  suited  for  histologic  specimens,  20  X  40  mm  for  larger 
sections.  The  thickness  should  be  of  an  average  of  0.12 — 0.15  mm;  thinner 
glasses  are  unnecessarily  difficult  to  clean,  while  thicker  glasses  will  not  admit 
the  use  of  the  oil-immersion  lens.  They  are  best  kept  in  a  covered  vessel,  con- 
taining 30%  alcohol.  The  cover-glass  is  taken  out  with  forceps  and  dried  with 
a  clean  cloth  before  using. 

Influence  of  the  Mounting 
Medium  on  the  Microscopic  Picture, 

Of  the  innumerable  media  recommended  for  mounting  we  are  interested  in 
just  three,  glycerine,  Canada  balsam  and  levulose.  Before  going  into  details, 
we  must  make  a  few  general  remarks  about  their  refractive  properties.  Our 
microscopic  preparations  possess  a  certain  refraction  index ;  if  they  should  be 
placed  in  a  medium  with  the  same  refraction  index,  all  structural  details  will  be 
lost,  will  be  made  invisible.  Before  being  stained,  the  entire  specimen  appears 
equally  illuminated.  Staining  will  bring  out  the  different  hues  in  elegant 
fashion.  If  we  desire  to  demonstrate  the  structural  details  in  the  unstained 
specimen,  we  must  select  a  medium  with  low  refractive  index,  hence  unstained 
specimens  are  best  to  examine  in  a  low  refractive  medium,  e.g.,  water,  which 
has  a  refractive  index  of  1.3.  It  is  different  with  stained  preparations,  where 
the  colors  will  not  appear  prominently  enough  in  a  low  refractive  medium ; 
here  it  is  better  to  select  a  medium  with  a  higher  index,  say  about  1.5. 

Glycerine. 

C3H5(OH)3,  a  thick  colorless  fluid,  mixing  with  water  and  alcohol  in  any 
proportion,  is  a  good  solvent  for  inorganic  and  organic  substances.  Its  re- 
fractive index  is  1.45.  It  is  used  a  great  deal  as  a  mounting  medium,  but  does* 
not  prevent  the  bleaching  of  the  stain  for  any  length  of  time.  Pure  glycerine, 
acting  hygroscopic,  will  shrink  the  specimen ;  it  is  therefore  advisable  to  use 
a  mixture  of  glycerine  and  water  first,  and  then  follow  with  pure  glycerine. 

Canada  Balsam. 

This  is  made  of  the  resin  of  several  North  American  fir-trees,  has  a  weak 
acid  reaction  and  is  soluble  in  ether,  benzol,  xylol  and  chloroform.  The  refrac- 
tive index  is  1.53.  After  the  acid  reaction  has  been  neutralized  by  potassium 
carbonate,  Canada  balsam  represents  about  the  best  mounting  material  for 
stained  specimens  known  to  this  day.  It  should  be  dissolved  in  chemically  pure 
xylol  sufficiently  to  allow  the  solution  to  drop  easily  and  be  kept  in  glasses  with 
a  surmounting,  closely  fitting  glass  top. 

Specimens  to  be  mounted  in  balsam  must  first  be  thoroughly  dehydrated 
by  absolute  alcohol  and  transferred  to  xylol.  The  slide  is  dried  on  its  under 
surface  and  around  the  specimen,  and  a  small  drop  of  balsam  is  dropped  on  the 
section,  which  latter  must  never  be  allowed  to  become  entirely  dry.  The  cover- 
glass  is  now  placed  in  the  manner  described  above. 


79 

Celloidin  sections  must  not  be  dehydrated  in  absolute  alcohol,  lest  the  cel- 
loidin  should  be  softened  too  much.  We  therefore  treat  it  with  up  to  95% 
alcohol,  after  which  the  small  amount  of  water  is  extracted  with  carbol  xylol, 
i.e.,  a  solution  of  1  part  of  phenol,  liquefied  by  heating,  in  3  parts  of  xylol. 
After  the  sections  have  become  entirely  transparent,  they  can  be  transferred 
to  xylol  and  are  then  ready  for  balsam. 

After  a  few  days  the  xylol  on  the  edge  of  the  cover-glass  will  have  evapo- 
rated sufficiently  to  make  a  strong  connection  between  cover-glass  and  slide, 
while  the  balsam  in  the  proximity  of  the  specimen  will  remain  liquid  for  months. 
It  is  to  be  recommended,  especially  in  dealing  with  delicate  stains  such  as 
Biondi  solution,  to  place  the  slide  in  the  thermostat  for  a  few  days  immediately 
after  it  has  been  finished. 

Levulose,  Fruit  Sugar. 

The  purest  form  is  found  on  the  market  in  form  of  light  yellow  crystals, 
readily  soluble  in  water  and  alcohol  to  form  a  yellowish  sirup ;  it  has  a  refrac- 
tive index  of  1.5. 

The  only  useful  preparation  is  the  crystallized  levulose,1  of  which  20  gms 
are  placed  in  a  balsam  glass  containing  15  cm3  of  water,  the  glass  to  be  kept  in 
the  paraffin  oven  overnight.  Again  we  dry  the  cover-glass  in  the  manner  de- 
scribed above,  trying,  however,  to  withdraw  any  excess  of  water  from  the 
specimen  itself  by  means  of  filter  paper.  A  small  drop  of  levulose  is  now  placed 
on  the  specimen,  which  is  left  uncovered  for  one  to  two  minutes.  The  cover- 
glass  is  now  adjusted  and  the  slide  kept  in  the  thermostat  for  several  hours  to 
allow  the  cover-glass  to  settle  evenly ;  lastly  we  enclose  with  cover-glass  cement. 
Levulose  is  an  excellent  mounting  medium,  possessing  the  advantage  over  bal- 
sam of  dispensing  with  alcohol  following  the  staining.  It  preserves  better  than 
glycerine,  being  inferior  to  balsam  in  this  respect. 


MENSURATION  AND    DRAWING  OF  MICROSCOPIC 

PREPARATIONS 

In  order  to  ascertain  the  actual  size  of  the  specimen  seen  in  our  micro- 
scopical image,  we  can  proceed  in  different  ways.  We  may  either  measure  the 
specimen  itself  by  the  aid  of  proper  devices  or  we  can  primarily  project  a  true 
picture  of  it  on  paper  by  means  of  the  drawing  apparatus,  measure  this  sec- 
ondarily and,  with  due  consideration  of  the  magnifying  power  used,  calculate 
the  true  size. 

Ocular  Micrometer. 

Apparatus  and  devices  for  the  measuring  of  microscopic  specimens  are 
called  micrometers.  They  are  constructed  after  various  principles  and  in  dif- 
ferent forms.  Simplest  and  most  efficient  for  our  purpose  is  the  ocular  microm- 
eter. It  consists  of  a  small  round  glass  plate,  provided  with  scale ;  the  scale 
is  5  mm  long  and  divided  into  50  equal  parts.  Special  eyepieces  are  necessary 
for  the  use  of  the  ocular  micrometer,  in  which  the  eye-lens  can  be  moved  toward 

1  C.  A.  F.  Kahlbaum,  Berlin.     Not  inexpensive. 


80 

and  away  from  the  collective  lens  by  means  of  a  draw-tube.  The  latter  is 
removed  and,  scale  down,  the  micrometer  is  placed  on  the  ocular  diaphragm. 
When  the  eye-lens  is  replaced,  we  will  thus  obtain  a  magnified  picture  of  the 
scale.  The  eyepiece  being  adjusted  in  the  barrel,  the  images  of  scale  and 
specimen  will  cover  each  other,  since  the  former  is  projected  through  the  eye- 
lens  in  the  same  plane  as  the  latter.  By  moving  the  specimen  we  can  place  any 
desired  field  under  the  scale  and  measure  how  many  of  the  subdivisions  on  the 
scale  it  will  cover.  Each  segment  of  the  scale  has  a  certain  value,  which  de- 
pends on  the  size  of  the  image  produced  at  the  level  of  the  ocular  diaphragm, 
viz.,  it  depends  on  the  focal  distance  of  the  objective.  The  more  powerful  the 
latter,  the  smaller  will  be  the  value  of  each  division  of  the  scale.  The  optician 
furnishes  tables  with  the  micrometer,  stating  the  values  for  the  different  ob- 
jectives in  a  sufficiently  accurate  manner,  so  that  we  simply  have  to  multiply 
the  number  of  subdivisions  with  the  value  stated  in  the  table.  It  goes  without 
saying  that  during  such  calculations  the  prescribed  barrel  length  must  be 
strictly  observed. 

Drawing  of 
Microscopic  Specimens. 

Of  the  significance  and  importance  of  drawing  we  have  already  spoken  in 
the  preface;  suffice  it  to  say  that  it  is  of  the  utmost  importance  that  the  be- 
ginner should  draw  his  specimens  repeatedly  and  in  an  exact  manner.  Most 
every  beginner  is  possessed  with  a  disliking  for  drawing,  which  we  can  often 
trace  to  an  underestimation  of  the  student's  own  faculties.  The  first  attempt, 
if  made  at  all,  is  generally  such  an  utter  failure,  that  the  student  becomes  dis- 
couraged and  fails  to  try  again.  For  this  reason  it  is  well  to  aid  the  first 
attempts  by  the  use  of  a  drawing  apparatus.  Noticing  how  the  picture  develops 
under  his  hands  by  the  simple  tracing  with  pencil,  he  will  acquire  the  ambition 
to  complete  the  sketch,  and  after  several  such  attempts  he  will  have  developed 
an  interest  and  pleasure  in  this  sort  of  work. 

Drawing  Apparatus. 

Of  the  various  makes  of  such  apparatus  we  prefer  the  simple  and  cheap 
Abbe  drawing  apparatus  (Fig.  30).  It  consists  of  two  metal  rings,  the  lower 
of  which  (r,)  is  fastened  centrically  with  three  screws  (schr)  upon  the  upper 
extremity  of  the  barrel;  the  other,  upper  ring  (r.2),  supports  the  apparatus 
proper  and  is  connected  with  the  first  ring  by  a  joint  (gel),  allowing  of  a  for- 
ward movement  of  the  apparatus.  Upon  the  upper  ring  we  find  a  small  glass 
cube,  enclosed  in  a  little  metal  drum  (tr),  which  is  open  on  the  side;  the  cube 
consists  of  two  prisms,  their  diagonal  planes  being  in  approximation.  The 
diagonal  plane  of  the  upper  prism  has  been  silvered  mirrorlike,  with  the  excep- 
tion of  a  small  central  pupil  of  about  2  mm  diameter.  In  front  a  horizontal 
arm  (a)  is  seen  emerging  from  the  upper  ring;  at  a  distance  of  7 — 8  cm  a 
rectangular  mirror  is  attached  to  this  arm,  which  can  be  rotated  on  a  hori- 
zontal axis.  After  the  apparatus  is  properly  attached,  the  mirror  is  adjusted 
at  an  angle  of  45°  to  the  arm,  and  now  the  observer,  looking  through  the 
opening  (o)  in  the  drum  and  thence  through  the  pupil  of  the  prism,  can  see 
the  microscopic  picture  and  at  the  same  time  the  point  of  a  pencil,  which  is 


81 

held  under  the  mirror,  both  pictures  uniting  in  the  eye,  thus  making  it  possible 
to  trace  the  contours  of  the  microscopic  picture. 

The  use  of  the  apparatus  is  not  quite  as  simple  as  it  may  at  first  appear 
to  be,  since  differences  in  light  between  the  two  images  -may  result  in  one  out- 
balancing the  other.  The  apparatus  being  thrown  back,  we  first  focus  our 
specimen  sharply,  and  close  the  iris  diaphragm;  the  point  of  the  pencil  must 
contrast  on  the  white  drawing  paper.  The  diaphragm  is  now  opened  slowly, 
until  specimen  and  pencil  point  appear  equally  distinct.  Should  the  drawing 
surface  be  too  light,  which  seldom  is  the  case,  smoked  glasses  should  be  inter- 
posed between  prism  and  mirror.  If  we  desire  correct  size,  we  must  be  sure  to 


echr 


FlG.  30. 
Drawing  Apparatus  after  Albe. 

have  the  drawing  surface  on  a  level  with  the  stage,  and  to  avoid  distortions  our 
mirror  must  be  kept  at  the  angle  of  45°.  Only  those  places  vertically  under 
the  mirror  should  be  drawn ;  if  larger  drawings  are  desired,  the  drawing  surface 
must  be  moved  accordingly. 

This  apparatus  serves  only  for  the  drawing  of  contours,  the  details  must 
be  worked  in  by  free  hand,  reading  from  the  microscopical  picture.  For  this 
purpose  we  may  use  pencils  .of  different  strengths  or,  better  still,  a  brush  and 
india  ink  or  colors.  The  drawing  pen  should  be  avoided,  as  it  will  impart  a 
certain  hardness  to  the  picture  which  is  foreign  to  the  specimen.  Of  the 
brushes  the  Japanese  are  the  best,  results  being  obtained  with  them  which  are 
even  more  delicate  than  could  be  produced  by  the  pen.  Of  the  stains  we  would 
recommend  the  Gouache  colors,  manufactured  by  Schoenfeld  &  Co.,  in  Diissel- 
dorf .  When  minute  details  are  desirable,  it  will  be  a  good  plan  to  draw  through 
a  simple  lens,  magnifying  two  to  three  times. 


Index 


Abbe's  condenser,  19 

Abbe's   drawing   apparatus,   80,   81 

Aberration,  chromatic,  7,  8,   12 

spherical,   7,    12,    15 
Acetic    acid,    29,    30,   35 
Achromatic  objective,  13,  15 
Acid  dyes,  54,  63 
Acid   fiichsin,  64,  67 

S.  M.  P.,  67 
Acidophilic,  54 
Adjective  staining,  53 
Adjustment,   coarse,   11,   23 
After-treatment,  28 

Aktiengesellschaft  fiir  Anilinfabrikation,  67 
Alcohol,  26,  28,  32,  35,  52 

shrinking  due  to,  33 
Alcohol-acetic  acid,  33 
Altman's  bioblasts,  64 
Altmaris  mixture,  31 
Amniotic   fluid,  25 
Anaesthetizing  of  animal,  27 
Angle,  14 

of  section,  44,  45 
Aniline  dyes,  58 
Aperture,  14,  15 
Aplanasia,   15 

Apochromatic  objective,  13 
Aqueous   humor,  25 
Artery  clamps,  76 
Artificial  stains,  58 
Azureosine,  67 

Barrel   of   microscope,   10,   11 

Base  of  microscope,   10 

Basic   dyes,   53,   54,   58 

Basophilic,  54 

Berlin  blue,  74 

Bichloride  of  mercury,  31,  48 

Biondi  solution,  67 

Bleu  de  Lyon,  64 

Blocking,  44 

Blood  serum,  25 

Blood,  staining  of,  65 

Blue  glue  mass,  74 

Body  of  microscope,   10 

Bordeaux  R.,  65 

Bull's  eye  condenser,  18 

Canada   balsam,    78 
Cannula,  75,  76 

introduction  of,  75 
Carbonic  acid,  27,  37 
Carmalum,   55 
Carmine,  54,  55 
Carminic   acid,  55 
Camay's  mixture,  33 
Carrier,    11 
Cedarwood  oil,  14 
Cell,  chemical  ingredients  of,  26 


Celloidin,  36,  46 

blocking,  46 

section  method,  46,  47 

staining,   52 
Cement,  77 

Changes  in  tissue  after  death,  24 
Chloroform,  27,  41 
Chop  method,  34,  35 
Chromatic  aberration,  7,  8,   12 
Chrome  hyperoxide,  29 
Chrome-osmio-acetic  acid,  30,  48 
Chrome    silver   method,   69,   70 

rapid,  70 
Chromic  acid,  29 
Clips,   10 

Coagulation  of  cellular  contents,  26 
Coarse  adjustment,  11,  23 
Coccus  cacti,  54 
Cochenille,  54 
Collective  lens,  9,  17 
Collodium,  45 
Color  bath,  49 

temperature   of,    50 
Color  lakes,  53 
Combined    system,   8 
Compensation   eyepiece,   17 
Compound  lens,  7 
Compound  microscope,  8,  9 
Concave  mirror,  19 
Condenser,   11,   12,    18,   19 

Abbe's,  19 

bull's   eye,   18 
Correction   lens,   12,   13 
Corrosive  sublimate,  31,  48 

chrome-osmium-acetic  acid,  32 

Mueller's  fluid-acetic  acid,  32 
Cover-glass,    13,    24,    77 

cement,  77 

size  of,  77 

Cresyl  violet  R.  B.,  62 
Crown   glass,  5,   7 
Curvature   radius,   7 
Cutting   instruments,   37 
Cutting  method,  34,  35 
Cutting  methods,  special,  47 
Cutting  of  paraffin  sections,  44 
Cylinder,   diaphragm,   20 

Decalcification,  48 
Dehydration,   41,   46 
Detailing  power,   15 
Dewar  bottle,  37,  38 
Diaphragm,  11,  17,  19,  20 

cylinder,   20 

iris,  19,  20 

lever,  20 

Differentiation  agents,  53 
Diffuse  staining,  53 
Dispersion,  5,  8 

83 


Dissociation   method,  34 
Distance,  visual,  7 
Drawing  apparatus,  80 
Drawing  of  specimen,  80 
Draw-tube,   11 
Durability  of  specimen,  76 

Elective  staining,  53 
Embedding,  36,  43 

frame,  43 
Eosine,  65 

Erlenmayer  flask,  67 
Ethyl  alcohol,  32 
Eye-lens,  9,  17 
Eyepiece,  9,  12,  17,  18 

marking  of,  18 

significance  of,  18 

Fat,  staining  of,  65 
Fixation,   after-treatment,  28 

amount   of   solution,   27 

duration  of,  28 

general  rules  for,  26 

reagents  for,  26,  62 

significance  of,  25 
Fixed  specimen,  cutting  of,  36 
Fleming's  solution,  30,  32 
Flint   glass,   5,    7,   8 
Fluor  spar,   13 
Focal   distance,  6,  7,  8 
Focus,  6 
Focussing,  22 
Foot  of  microscope,  10 
Formalin,  26,  28,  29,  33,  48 

alcohol,   34 

Mueller's  fluid,  33 
Freezing  cylinder,  37,  38 
Freezing  microtome,  38,  39 
Fresh  specimen,  cutting  of,  36 
Front  lens,  12 
Frozen    section,   36,  37 

method,  37,  47 

staining  of,  51 
Fuchsin,    58 

Gentian  violet,  59 
Giemsa  solution,  67 
Glauber  salt,  29,  31,  48,  76 
Glue  mass,  74 
Glycerine,  35,  78 

for  mounting,   78 
Gold  chloride,  72 

formic  acid  method,  73 

Gram  method,  73 

lemon  juice,  72 
Oolgi  method,  69 
Gouache  colors,  81 
Gram's   method,   59 
Green  chrome  oxide,  29 

Haemalum,   56,   65 
Haematin,  56 
Haematoxylin,  56 

iron-alum,  57 

phospho-molybdic   acid,  57 

potassium-dichromate,  57 
Heat,  a  fixing  agent,'  26 
Heidenhain  staining,  57 
Hermann's  fluid,  32 

Heterogeneous  staining  mixtures,  65,  66 
High  power,  13 


Hoechster  Farhwerke,  59 
Homogeneous   immersion,   14 
Homogeneous   staining  mixtures,  65,  66 
Hifi/tjhen  eyepiece,   17,   18 
Hydrochloric  acid,  32,  35,  63 
Hydrocyanic  acid,  27 
Hydrogen   peroxide,  30 
Hypertonic  fluids,  25 
Hypodermatic   method   of  staining,  50 
Hypotonic   fluids,  25 

Illumination,  18,  19,  24 
Image,  6 

planatic,  15 

Immersion  objective,  13 
Impregnation,  68,  69 

after-treatment,  70 

duration  of,  70 

with  gold  salts,  72 

with   silver   salts,   69 
Indifferent  stains,  65 
Indigcarmin,  58 
Indigo,   57 

Injection   apparatus,  60,  61,  74 
Injection  material,  73,  74 
Injection  method,  73,  74 
Injection  syringe,  74,  75 
Injection  technique,   75 
Intercellular  substance,  26 
Intravenous  method  of  staining,  50 
lodin,   31,  35 
Iris  diaphragm,   19,  20 
Iron-haematoxylin,  56 
Isolation-agents,  34 
Isotonic  fluids,  25 

Jena   glass,   67 

Joint  of  microscope,  10 

Lakes,  53 
Lauth'x   violet,   59 
Leitz  microscopes,  21 
Lens,  5 

classification,  5 

collective,  9,   17 

composition  of,  5 

compound,    7 

correction,    12 

eye,  9,  17 

negative,  5 

positive,  5 
Levulose,  79 
Light   green,   64 
Light,  source  of,  20,  21 
Living  object,  observation  of,  25 
Low   power,    13 
Lump    staining,   51 
Lyon   blue,   64 

Maceration,  34,  35 
Magnifying,  7,  9,  12 
Maximum   of  staining,   52 
Media  for  preservation,  25 
Meniscus,  5,  6 
Mensuration,   79 
Metachromasia,  54,  62 
Metachromatic  staining,  54 
Metal  oxides,  53 
Metals  for  impregnation,  68 
Metals,   heavy,  26,  28 
Methylene  azur,  62 


85 


Methylene   blue,   59,   60 
Methyl  green,  59 

N.  M.  P.,  67 
Methyl  violet,  59 
Micrometer,  79 

screw,   11,   12,  31,  23 
Microscope,  5,  23 

care  of,  22 

compound,  8,  9 

description  of,  9,  10 

illustration   (Fig.  8),  10 

prices  of,  22 

procuring  of,  21 

rules  for  use  of,  21 
Microtechnique,   definition   of,   24 

general,  24 
Microtome,  37,  44 
Mineral   acid,  26 
Mirror,  11,  19 

concave,  19 

plane,    19 
Mixtures  of  stains,  65 

heterogeneous,  65,  66 

homogeneous,  65,  66 
Monobrome-naphthalin,   14,  15 
Mordants,  53 
Morphine,  27 
Mounting,  45,  71,  76,  77,  78 

media,   77,  78 

of  impregnated  specimens,  71 
Mueller's  fluid,  31 
Multiple  staining,  52,  65 

Nerve-sheath,  staining  of,  65 
Nitric  acid,  28,  31,  35,  48 
Nosepiece,    11,    12 
Nuclear   dyes,    58 
Nuclear   staining,  60 
Numerical  aperture,  14 

Object  distance,  13 

Object  glass.     See  Objective 

Objective,  9,  12 

achromatic,   13,   15 

apochromatic,   13 

cleaning  of,  22 

illustration   (Figs.  10,  11,  12),  16 

marking  of,  15 
Ocular  (eyepiece),  9,  17 
Ocular  micrometer,   79 
Oil  immersion  objective,  13,  14,  15,  23 
Oil  of  cloves,  45 
Optic  axis,  12,  13 
Optic  system,  8 
Optimum  of  staining,  52 
Orange  G.,  64 
Orange  G.   M.   P.,  67 
Orthochromatic   staining,  54 
Osmic  acid,  29,  30,  31 
Osmium   tetroxide,  29 

Paracarmine,   55 
Paraffin,  36,  41,  42 

embedding,    43 

melting-point,  42 

preparatory  media,  41 

section  method,  41,  47 

staining   of  sections,   52 
Penetrating   power,  27 
Picric   acid,   29,  64,  65 
Picrocarmin,    66 


Picrofuchsin,   66 

Picro-indigcarmin,  66 

Picte-t,  37 

Planatic  image,   15 

Plane   mirror,   19 

Platinum  chloride,  28,  32 

Platinum  chloride-osmio-acetic  acid,  32 

Post-mortem  specimens,  27 

Potassium  bichromate,  30,  31 

Potassium  bichromate-osmic  acid,  31 

Potassium  cyanide,  29 

Potassium  hydroxide,  35 

Power  of  objective,  13 

Preservation,    media    for,    25 

method  of,  24,  25,  46 
Primary  lens,  8 
Prism,  metal,  11 
Progressive  staining,  52 
Protection  of   hands,   61 
Pyrogallol  method,   71 

Ramsdcn  eyepiece,  17 
Rays,  course  of,  8 
Razor,  37 
Red  glue  mass,  74 
Reduction,  61,  68 
Reflected   light,   18 
Refraction,   5 

index  of,  7,  13 
Refractive   errors,   12 
Regressive  staining,  52 
Reichert,  21 
Resorcin   fuchsin,  63 
Retrogressive.     See  Regressive 
Ringer's  fluid,  25,  51,  59,  60,'  61 

Safranine,   63 

Saline,  solution  of,  25 

Saturation  with  celloidin,  46 

Scarlet  R.,  65 

Schanze,  38 

Schoenfeld  &  Co.,  81 

Section,   24 

method  of,  24 

preparation  of,  34,  47 

staining  of,  51 

thickness  of,  40 

treatment   of,   40 
Sedimentation,    35 
Seibert,  21 

Silver-ammonia  method,  71 
Silvering,  69 

Silver   nitrate,   for  impregnation,   69 
Simple    staining,    52 
Simultaneous    mordant    dyeing,    53 
Simultaneous   staining,   52 
Size  of  specimen,  27 
Sketching,  80 
Slide,  24 

Sodium  sulphate,  31,  32 
Soluble  Berlin  blue,  74 
Solvents  of  dye,  49 
Spherical   aberration,   7,   12,   15 
Stabilit,  47 

Stage  of  microscope,  10 
Staining,  24 

adjective,   53 

diffuse,  53 

elective,  53 

method  of,  24,  48 

mixtures,   65 


86 


Staining  of  celloidin,  52 
of  frozen  specimen,  51 
of  living  specimen,  50 
of   paraffin    specimen,    52 
of   preserved   specimen,   51 
of  surviving  specimen,  50 
progressive,  52 
regressive,  52 
simultaneous,    52 
substantive,  53 
succedaneous,  52 
Stains,  49 

artificial,  58 
concentration  of,  49 
indifferent,   65 
in  mixture,  65 
of  animal  origin,  54 
of  vegetable  origin,  55 
Sublimate-nitric   acid,   48 
Substantive  staining,  53 
Succedaneous  staining,  52 
Succedaneous  mordant  dyeing,  53 
Successive.     See  Succedaneous 
Sudan  III.  65 


Tannic  acid,  26,  58 
Tar-dyes,  58 
Thermos  bottle,  37,  38 
Thermostat,  42      , 
Thionine,   59 

Transmitted  light,  19,  24 
Trichlor-acetic  acid,  29,  31 
Tube  length,  9 

Vital  staining,  50 

Vital  staining  of  nerves,  60 

Voigtlander,  21 

Wash-glass,   28 

Water  as  immersion  medium,  14,  15 

Winkel,  21 

Wooden  slide,  71 

Xylol,    52 

Zeiss,  21 
Zenker's  fluid,  32 


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